Application for an Extension of the Determination of Nonregulated

Status for Insect-Resistant, Glufosinate Ammonium-Tolerant Cotton 

(08-340-01p): 

Transformation Event T303-3

OECD Unique Identifier BCS-GHØØ3-6 

Submitted by : 

________________________________

Michael Weeks, M.S.

Registration and Compliance Specialist

Regulatory Affairs – BioScience

Bayer CropScience LP

2 T.W. Alexander Drive

P.O. Box 12014

Research Triangle Park, NC 27709

Telephone: 919-549-2119

e-mail: michael.weeks@bayer.com

Contributors:

Isabelle Coats

Penny Hunst

Mark Rinehardt

January 30, 2012

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RELEASE OF INFORMATION

The information in this petition is being submitted by Bayer CropScience LP for review 

by USDA as part of the regulatory process. By submitting this information, Bayer 

CropScience LP does not authorize its release to any third party except to the extent it is

requested under the Freedom of Information Act (FOIA), 5 U.S.C., Section 552 and 7 

CFR 1, covering all or some of this information. Except in accordance with FOIA, Bayer 

CropScience LP does not authorize the release, publication or other distribution of this 

information without Bayer CropScience’s prior notice and consent.

©2011 Bayer CropScience. All rights reserved.

This document is property of Bayer CropScience and is for use by the regulatory authority to 

which it has been submitted, and only in support of actions requested by Bayer CropScience. All 

data and information herein must not be used, reproduced or disclosed to third parties without the 

written consent of Bayer CropScience

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CERTIFICATION

The undersigned certifies, that to the best knowledge and belief of the undersigned, this 

petition includes all information and views on which to base a determination, and that it 

includes relevant data and information known to the petitioner which is unfavorable to 

the petition.

 

Michael Weeks, M.S.

Registration and Compliance Specialist

Regulatory Affairs – BioScience

Bayer CropScience LP

2 T.W. Alexander Drive

P.O. Box 12014

Research Triangle Park, NC 27709

Telephone: 919-549-2119 

e-mail: michael.weeks@bayer.com

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SUMMARY

Bayer CropScience requests a determination from USDA APHIS that insect-resistant, 

glufosinate ammonium tolerant cotton event T303-3 and any progeny derived from 

crosses of this event with traditional cotton varieties, and any progeny derived from 

crosses of this event with transgenic cotton varieties that have also received a 

determination of nonregulated status, no longer be considered regulated articles under 7 

CFR Part 340, and that APHIS consider this document as an extension to petition 08-

340-01p. The subject of petition 08-340-01p, TwinLinkTM Cotton, received a 

determination of non-regulated status on September 23, 2011.

Gossypium hirsutum transformation event T303-3 was produced by Agrobacteriummediated transformation utilizing vector pTDL004 containing a cry1Ab gene construct, 

encoding insect-resistance, and the bar gene as a selectable marker conferring 

tolerance to glufosinate ammonium herbicides. The antecedent organism, cotton event 

T304-40 as described in petition 08-340-01p, was also generated through 

Agrobacterium-mediated transformation utilizing a slightly different vector (pTDL008). 

Both events produce the same insecticidal crystal protein (ICP) Cry1Ab (expression 

product of the cry1Ab gene) and PAT protein (expression product of the bar gene). 

Molecular analysis has demonstrated that event T303-3 contains one stably integrated

intact copy of the cry1Ab gene and bar gene cassettes. Evaluation of the Cry1Ab 

protein expressed by event T303-3 was found to be comparable to that expressed by 

event T304-40 as well as that expressed in E. coli for the purposes of protein safety 

assessment in petition 08-340-01p.

Agronomic evaluations demonstrated that no differences in morphology, disease or pest 

susceptibility were observed when compared to other cultivated cotton varieties. No 

adverse effects on beneficial organisms were found. Composition analysis of raw 

agricultural commodities produced by event T303-3 was found to be substantially 

equivalent to that of its non-transgenic counterpart.

Based on all analyses performed, event T303-3 was found to be comparable to the 

antecedent organism, event T304-40.

A comparison of characteristics of events T303-3 and T304-40 is summarized in Table 1 

and is discussed in the appropriate sections of the petition.

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Table 1. Comparison of events T304-40 and T303-3

Characteristic T303-3 T304-40

Crop Cotton Cotton

Recipient Organism Gossypium hirsutum Gossypium hirsutum

Parent Line Coker 315 Coker 315

Transformation Method Agrobacterium-mediated Agrobacterium-mediated

Trait Insect-resistant, herbicidetolerantInsect-resistant, herbicidetolerant

Gene Products Cry1Ab and PAT Cry1Ab and PAT

Vector pTDL004 pTDL008

Gene/Donor cry1Ab/Bacillus 

thuringiensis berliner 1715

cry1Ab/Bacillus 

thuringiensis berliner 1715

Promoter/Donor P35S2/Cauliflower Mosaic 

Virus promoter

Ps7s7/Subterranean Clover 

Stunt Virus promoter

Terminator/Donor 3’ me1/3’ untranslated 

region of NADP-malic 

enzyme gene of Flavia 

bidentis

3’ me1/3’ untranslated 

region of NADP-malic 

enzyme gene of Flavia 

bidentis

Gene/Donor Phosphinothricin 

acetyltransferase 

(bar)/Streptomyces 

hygroscopicus

Phosphinothricin 

acetyltransferase 

(bar)/Streptomyces 

hygroscopicus

Promoter/Donor P35S3/Cauliflower Mosaic 

Virus promoter

P35S3/Cauliflower Mosaic 

Virus promoter

Terminator/Donor 3’ nos/3’ untranslated 

region of nopaline synthase 

gene from T-DNA of pTiT37

3’ nos/3’ untranslated 

region of nopaline synthase 

gene from T-DNA of pTiT37

Molecular Weight Cry1Ab ~69 kDA ~69 kDA

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ACRONYMS AND SCIENTIFIC TERMS

a.i. Active ingredient

A Acre

ADF Acid Detergent Fiber

ANOVA Analysis of Variance

BC Backcross

BCS Bayer CropScience

bp Base pair

Bt Bacillus thuringiensis

cry1Ab Insecticidal crystal protein gene from Bacillus thuringiensis

Cry1Ab Insecticidal crystal protein expressed from the cry1Ab gene

d.w. Dry weight

F1, F2, etc Breeding generations denoting product of pollinations and 

subsequent self-pollinations following the cross of 

transformation event with conventional germplasm

ELISA Enzyme-Linked Immunosorbent Assay

FWS U.S. Fish & Wildlife Service

g Gram

g Earth’s gravitational acceleration (1 g = 3,530,394 cm / minute2)

ICP Insecticidal crystal protein

kDa Kilodalton

lb US pound

LB Left border of T-DNA

μg Microgram

MQ Milli-Q water

MW Molecular Weight

NDF Neutral Detergent Fiber

ng Nanogram

ORF Open Reading Frame

PAT/bar Phosphinothricin Acetyl-Transferase as expressed by the bar

gene

pg Picogram

RB Right border of T-DNA

SD Standard Deviation

T1, T2, etc Breeding generations denoting self-pollinations of the T0 plant

(transformation event)

T-DNA Transfer DNA from Agrobacterium tumefaciens

TwinLink Insect-resistant, herbicide-tolerant stacked cotton product 

(events T304-40 x GHB119)

U.S. United States

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TABLE OF CONTENTS

Title Page............................................................................................. 1

RELEASE OF INFORMATION.................................................................. 2

CERTIFICATION.................................................................................... 3

SUMMARY............................................................................................ 4

ACRONYMS AND SCIENTIFIC TERMS...................................................... 6

TABLE OF CONTENTS........................................................................... 7

LIST OF TABLES.................................................................................... 9

LIST OF FIGURES.................................................................................. 10

I. RATIONALE FOR NONREGULATED STATUS..................... 11

II. THE COTTON FAMILY....................................................... 12

III. DEVELOPMENT OF EVENT T303-3..................................... 13

 III.A. The Transformation System................................................. 15

 III.B. The Parental Line............................................................... 15

 III.C. Breeding History................................................................ 15

 III.D. Generations Used for Analyses............................................. 17

IV. GENETIC MATERIAL USED FOR TRANSFORMATION........... 18

 IV.A. Construction of Plasmid pTDL004.......................................... 18

 IV.B. Description of Genetic Elements and Regulatory Sequences...... 19

 IV.C. Identity and Source of Genetic Material................................... 19

V. GENETIC CHARACTERIZATION OF EVENT T303-3............... 20

 V.A. Insert Characterization........................................................ 20

 V.A.1. Southern Blot Analyses....................................................... 20

 V.A.2. DNA Sequencing............................................................... 24

 V.B. Open Reading Frames and Associated Regulatory Regions....... 25

 V.C. Confirmation of the Absence of Vector Backbone..................... 25

 V.D. Mendelian Inheritance......................................................... 31

 V.E. Conclusions...................................................................... 31

VI. CHARACTERIZATION OF THE INTRODUCED PROTEINS...... 32

 VI.A. Cry1Ab Protein.................................................................. 32

 VI.A.1. History and Background...................................................... 32

 VI.A.2 Characterization of the Cry1Ab Protein................................... 32

 VI.B. The Pat/bar Protein............................................................ 32

 VI.C. Protein Safety.................................................................... 32

 VI.D. Expression of Introduced Proteins.......................................... 33

 VI.E. Comparison of Cry1Ab Protein from Events T303-3 and T304-40. 35

VII. AGRONOMIC AND PHENOTYPIC EVALUATION................... 40

 VII.A. History of Field Activities...................................................... 40

 VII.B. Agronomic and Phenotypic Characteristics Evaluated............... 41

 VII.B.1. Agronomic and Plant Product Characteristics........................... 42

 VII.B.2. Assessment of Phenotype by Efficacy Against Insect Pests........ 45

 VII.C. Disease and Pest Characteristics.......................................... 46

 VII.D. Composition Analysis......................................................... 46

 VII.D.1. Proximate Analysis............................................................. 47

 VII.D.2. Anti-Nutrient Analysis.......................................................... 47

 VII.E. Conclusion........................................................................ 48

VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC 

PRACTICES....................................................................... 49

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 VIII.A. Potential for Gene Transfer................................................... 49

 VIII.B. Weediness Potential............................................................ 50

 VIII.C. Effects on Non-Target Organisms.......................................... 50

 VIII.D. Effects on Endangered Species............................................. 50

 VIII.E. Indirect Effects on Other Agricultural Products.......................... 51

IX. STATEMENT OF GROUNDS UNFAVORABLE........................ 52

X. REFERENCES.................................................................. 53

Appendix 1. Field Trial Termination Reports 2001-2006........................... 57

 

Appendix 2. Materials and Methods...................................................... 87

 2.A. Insert Characterization........................................................ 88

 2.B. Confirmation of Absence of Vector Backbone.......................... 89

 2.C. Protein (Cry1Ab and PAT) Expression in Raw Agricultural 

Commodity....................................................................... 91 

 2.D. Protein Characterization...................................................... 92

 2.E. Composition Analysis.......................................................... 93

Appendix 3. Characterization of Event T303-3........................................ 96

 3.A. Insert Characterization......................................................... 97

 3.B. Confirmation of Absence of Vector Backbone........................... 100

 3.C. Composition Analysis.......................................................... 103

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LIST OF TABLES

1. Comparison of events T304-40 and T303-3............................................. 5

2. Generations and backgrounds used for analysis.................................... 17

3. Genetic Elements of T-DNA from pTDL004.............................................. 19

4. Expected and Obtained Hybridization Fragments of Event T303-3............. 21

5. Mendelian segregation of cotton event T303-3....................................... 31

6. Cry1Ab protein levels in event T303-3 fuzzy seed.................................. 34

7. PAT/bar protein levels in event T303-3 fuzzy seed................................. 35

8. Field trial locations for evaluation of agronomic and phenotypic 

characteristics.................................................................................

40

9. Agronomic characteristics evaluated in field trials................................... 41

10. Yield and fiber quality characteristics evaluated in field trials..................... 41

11. Phenotypic (efficacy) characteristics evaluated in field trials..................... 41

12. Parameters assessed by year and location........................................... 42

13. Summary of agronomic data from 2005 field trials.................................. 42

14. Summary of agronomic data from 2006 field trials.................................. 43

15. Summary of plant product characteristics from 2005 field trials.................. 44

16. Summary of plant product characteristics from 2006 field trials.................. 45

17. Summary of phenotypic characteristics by efficacy against target pests...... 46

18. Analyses performed on cottonseed of event T303-3 and its non-transgenic 

counterpart..................................................................................... 47

19. Mean proximate composition in cottonseed of event T303-3..................... 47

20. Mean anti-nutrient composition in cottonseed of event T303-3.................. 48

21. Analytical methods used for compositional analysis................................ 95

22. Event T303-3 insert verification – probe information................................ 97

23. Event T303-3 vector backbone analysis – probe information..................... 100

24. Expected and obtained hybridization fragments with overlapping vector 

backbone probe templates................................................................. 101 

25. Expected and obtained hybridization fragments using T-DNA probe........... 102

26. Amino acid composition in cottonseed of event T303-3............................ 103

27. Fatty acid composition in cottonseed of event T303-3.............................. 104

28. Cyclopropenoid fatty acid composition in cottonseed of event T303-3........ 104

29. Mineral and tocopherol composition in cottonseed of event T303-3............ 105

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LIST OF FIGURES

1. Schematic of the development process of event T303-3 cotton................ 14

2. Breeding diagram for event T303-3 cotton........................................... 16

3. Vector map of pTDL004................................................................... 18

4. Event T303-3 insert organization by probe hybridization sizes................. 22

5. Hybridization results utilizing cry1Ab, bar, and T-DNA probes.................. 23

6. Insert organization of event T303-3.................................................... 24

7. Schematic drawing of probe strategy for vector backbone detection......... 27

8. Event T303-3 verification of absence of vector backbone – Southern blot 

results.......................................................................................... 29 

9. Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein 

isolated from events T303-3 and T304-40............................................ 36 

10. Comparison of trypsin-digested and thrombin-engineered Cry1Ab from 

E.coli to Cry1Ab isolated from event T304-40....................................... 38 

11. Event T303-3 insert verification – Southern blot results........................... 98

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I. RATIONALE FOR NONREGULATED STATUS

The United States Department of Agriculture (UDSA) Animal and Plant Health Inspection 

Services (APHIS) is responsible for protection of the US agricultural infrastructure 

against noxious pests and weeds. Under the Plant Protection Act (7 USC § 7701-7772) 

APHIS considers plants altered or produced by genetic engineering as restricted articles 

under 7 CFR 340 which cannot be released into the environment without appropriate 

approvals. APHIS provides that petitions may be filed under 7 CFR §340.6 to evaluate 

data to determine that a particular regulated article does not present a plant pest risk. 

Should APHIS determine that the submitted article does not present a plant pest risk; the 

article may be deregulated and released without further restrictions. 

This petition serves an application for an Extension of the Petition for Determination of 

Nonregulated Status for Insect-Resistant, Glufosinate Ammonium-Tolerant cotton: 

TwinLinkTM Cotton (events T304-40 x GHB119). The petition for TwinLink Cotton (08-

340-01p) received a determination of non-regulated status on September 23, 2011. 

Event T303-3 expresses the same Cry1Ab and PAT/bar proteins as event T304-40 and 

therefore there are no changes in the rationale from petition 08-340-01p entitled “Petition 

for Determination of Nonregulated Status for Insect-Resistant and Glufosinate 

Ammonium-Tolerant Cotton: TwinLinkTM Cotton (events T304-40 x GHB119)”. 

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II. THE COTTON FAMILY

The genus Gossypium is classified under the tribe Gossypiae, family Malvaceae, 

subfamily Malvoideae.

The OECD consensus document on cotton biology (OECD, 2008) provides information 

pertaining to the following aspects of cotton biology:

• Taxonomy, morphology and uses 

• Centers of origin of the species and domestication

• Agronomic practices

• Reproductive biology and dispersal

• Genetics and hybridization

• Interactions with other organisms

• Human health and biosafety

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III. DEVELOPMENT OF EVENT T303-3 

The T303-3 event was obtained using the same transformation system (Agrobacteriummediated) as was used to obtain the antecedent organism, event T304-40 (USDA, 

2008). The parental line used, Coker 315, was the same for event T304-40. A 

schematic of the development process of event T303-3 is shown in Figure 1.

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Figure 1. Schematic of the development process of event T303-3 cotton

cry1Ab gene (encoding the lepidopteran insecticidal crystal protein Cry1Ab)

bar (encoding the Phosphinothricin Acetyl-Transferase, PAT)

Assembly of plasmid pTDL004 containing cry1Ab and bar gene cassettes

Agrobacterium-mediated transfer of T-DNA from pTDL004

into cotton cells via co-cultivation

Selection of transformation events based on tolerance to 

glufosinate ammonium herbicide

Regeneration of T0 cotton plants

Evaluation of transformed plants for agronomic performance

and insect efficacy against target insects

Selfing and continued evaluation of agronomic

performance and insect efficacy

Selection of homozygous plants;

backcrossing and crossing for product development

Field and laboratory studies to support product

registration

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III.A. The Transformation System

EventsT304-40 and T303-3 were both generated through Agrobacterium-mediated gene 

transfer of the T-DNAs from pTDL008 and pTDL004, respectively. Each T0 plant was 

crossed with conventional cotton in order to obtain homozygous and stable lines. 

Agrobacterium-mediated gene transfer of a plasmid results in the transfer of the DNA 

fragment between the T-DNA border repeats to the plant genome. The left and right 

border repeats of A. tumefaciens are also inserted in the individual events. Even though 

some of the regulatory elements used in the transformation process were derived from 

A. tumefaciens, a known plant pathogen, the genes that cause crown gall disease were 

not part of the T-DNA, and therefore were not incorporated into the recipient plant 

(Deblaere et al., 1985). Section V.C. summarizes the analysis to show the absence of 

vector backbone in event T303-3.

III.B. The Parental Line

The parental line, Coker 315, is an older commercial variety of upland cotton 

(Gossypium hirsutum) which is no longer commercially cultivated. However, Coker 315 

is well suited for transformation due to its capacity for regeneration from tissue culture.

III.C. Breeding History

The T0 transformation event of Coker 315 was both self-pollinated and cross-pollinated 

with conventional cotton to produce T1 and F1 lines, respectively. The T1 and F1 lines 

then underwent a series of self- and cross-pollinations as well as backcrosses to 

produce stable lines for the various assessments outlined in this petition. See Figure 2 

for a schematic of the crossing scheme utilized. 

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Figure 2. Breeding diagram for event T303-3 cotton

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III.D. Generations Used for Analyses

Table 2 provides the generation and genetic background of event T303-3 used for each 

analyses presented in this petition.

Table 2. Generations and backgrounds used for analysis

Analysis Conducted Generation 

Insert Characterization by Southern Blot 

Analysis

BC2F2

DNA Sequencing BC2F2

ORF Analysis BC2F2

Absence of Vector Backbone BC2F3

Mendelian Inheritance T1, T2, F2, BC3F3

Protein Expression T4

Comparison of Cry1Ab proteins from 

events T303-3 and T304-40

T4 

2005 Agronomic and Phenotypic 

Evaluation in Field

T4

2006 Agronomic and Phenotypic 

Evaluation in Field

T5

Composition Analysis T4

 

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IV. GENETIC MATERIAL USED FOR TRANSFORMATION

IV.A. Construction of Plasmid pTDL004

The vector pTDL004 is a derivative of the vector pGSV20 in which the bar gene cassette 

coding for the phosphinothricin acetyltransferase protein of Streptomyces hygroscopicus

(Thompson et al., 1987) was inserted together with the cry1Ab gene cassette encoding 

the Cry1Ab crystal protein of Bacillus thuringiensis subsp. berliner (Höfte et al., 1986). A 

map of plasmid pTDL004 is provided in Figure 3. 

Figure 3. Vector map of pTDL004

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IV.B. Description of Genetic Elements and Regulatory Sequences

Regulatory sequences in the T-DNA plasmid of vector pTDL004 include two copies of 

the P35s promoter sequence (P35s3 and P35s2), 3’nos terminator sequence, 3’me1 

terminator sequence, and the 5’e1 leader sequence. Genetic elements in the T-DNA 

plasmid of vector pTDL004 are the bar and cry1Ab (Cry1Ab5PGS3a) gene sequences. 

Also included in the T-DNA of vector pTDL004 are the left and right border repeats (LB 

and RB, respectively) from the pTDL004 vector. Other genetic elements of vector 

pTDL004 occupying nucleotide positions from 5176 bp to 13904 bp (ORI pVS1 and ORI 

colE1) should not be inserted into the plant genome during the transformation process. 

For event T303-3 cotton, absence of these genetic elements (i.e., vector backbone) was

confirmed (Section V.C.). 

IV.C. Identity and Source of Genetic Material

Nucleotide positions within the plasmid pTDL004, orientation, identity and source of 

genetic elements and regulatory sequences are provided in Table 3. 

Table 3. Genetic elements of T-DNA from pTDL004

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V. GENETIC CHARACTERIZATION OF EVENT T303-3 

Genetic characterization of event T303-3 demonstrated that one copy of the cry1Ab

gene cassette and one copy of the bar gene cassette were inserted as a result of the 

transformation. The DNA sequence of the pre-insertion locus as well as the insert was 

determined. The 5’ and 3’ flanking sequences of the insertion site of event T303-3 and 

the pre-insertion locus of wild type G. hirsutum were determined and no rearrangements 

of genomic DNA were found. Further investigation confirmed the absence of any 

elements of the vector backbone. 

V.A Insert Characterization

V.A.1. Southern Blot Analyses

Southern blot analyses were used to characterize the insert present in event T303-3 

(Habex et al., 2007). To this end, several identically prepared membranes were loaded 

with samples of event T303-3 genomic DNA digested with nine different restriction 

enzymes. The appropriate positive control samples (genomic DNA from non-transgenic 

cotton supplemented with one copy pTDL004 DNA digested with NotI) and negative

controls (genomic DNA from non-transgenic cotton) were also prepared and included on 

the membranes. The blots were sequentially hybridized with different probes, either

designed to hybridize to a specific component of the cry1Ab or bar gene cassettes or to

the complete T-DNA. A detailed description of the materials and methods employed for

the insert characterization of event T303-3 are provided in Appendix 2.A. A summary of 

the restriction enzymes and T-DNA probes used as well as expected and obtained 

hybridization fragments is provided in Table 4. 

 

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Table 4. Expected and Obtained Hybridization Fragments of Event T303-3 

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Based on the obtained hybridization results, the organization of the inserted transgenic 

sequences of event T303-3 were determined and demonstrated that the insert of event 

T303-3 has one complete copy of each the cry1Ab and bar gene cassettes. Figure 4 

shows a schematic presentation of the insert organization deduced by hybridization 

fragment sizes. Restriction enzymes and probes as well as the expected hybridization 

fragments are indicated. 

Figure 4. Event T303-3 insert organization by probe hybridization sizes

Figure 5 shows the results of the Southern analyses utilizing the cry1Ab, bar, and 

complete T-DNA probes. Additional results for all Southern analyses utilized for insert 

characterization are presented in Appendix 3.A.

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Figure 5. Hybridization results utilizing cry1Ab, bar and T-DNA probes

 cry1Ab probe bar probe

 Complete T-DNA probe

Loading Sequence- All Probes Shown

Lane 2: G. hirsutum event T303-3 – BamHI digested

Lane 3: G. hirsutum event T303-3 – BglII digested

Lane 4: G. hirsutum event T303-3 – SacI digested

Lane 5: G. hirsutum event T303-3 – KpnI digested

Lane 6: G. hirsutum event T303-3 – NcoI digested

Lane 7: G. hirsutum event T303-3 – PvuI digested

Lane 8: G. hirsutum event T303-3 – XbaI digested

Lane 9: G. hirsutum event T303-3 – EcoRV digested

Lane 10: G. hirsutum event T303-3 – NotI digested

Lane 11: G. hirsutum wild type – NotI digested

Lane 12: G. hirsutum wild type + 1 copy pTDL004 –NotI 

digested

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V.A.2. DNA Sequencing

DNA sequencing of the transgenic locus confirmed the results of the characterization by 

Southern analyses which indicated that the inserted sequence of event T303-3 has one 

intact copy of each the cry1Ab and bar gene cassettes (Moens et al., 2007). DNA 

sequencing also confirmed the arrangement of the inserted DNA elements as deduced 

from Southern analyses (Figure 6). As shown below, the insertion resulted in 

rearrangements of genetic elements of the T-DNA from pTDL004. However, each of the 

gene cassettes were inserted as complete and single copies

Figure 6. Insert organization of event T303-3 

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The pre-insertion locus, the inserted sequences and the flanking sequences were 

determined by DNA sequence analysis of event T303-3 and non-transgenic G. hirsutum

(Moens et al., 2007). The results showed a target site deletion of 1184 bp. The putative 

function of the deleted sequence was not explored; however agronomic and composition 

analyses (Section VII) indicate that event T303-3 is comparable to other G. hirsutum

varieties. 

Event T303-3 flanking sequences of 776 bp at the 5’ and 251 bp at the 3’ junctions, 

respectively, were determined and were shown to be identical to sequences at the preinsertion locus of non-transformed G. hirsutum. 

V.B. Open Reading Frames and Associated Regulatory Regions

During the transformation process, the T-DNA from plasmid pTDL004 underwent some 

rearrangements creating several new junctions as shown in Figure 6. For these regions, 

an investigation was conducted as to whether any novel unexpected proteins could be 

expressed due to the insertion and rearrangement of the gene cassettes (De Pestel, 

2008). 

Open reading frame (ORF) analysis and gene search tools were applied to predict the 

presence of any potential newly created coding sequences in the junction regions. 

Seven newly created ORFs were identified and characterized. Bioinformatics tools were 

applied to look for regulatory elements needed for transcription and translation of any 

potential new ORFs. Four putative promoter regions were identified, however, none of 

them were found to be in a configuration likely to initiate transcription of the predicted 

ORFs. Thus, it was concluded that the expression of a new proteins from the seven

predicted ORFs was highly unlikely as the regulatory elements necessary to initiate 

transcription and translation were not present.

In addition, the putative amino acid sequences of the seven ORFs were also compared 

with sequences of known allergens and toxins using publically available protein

databases Uniprot-Swissprot (release 55.6, 2008), Uniprot-TrEMBL (release 37.8, 

2008), PDB (release 2007), DAD (release 37.8, 2007), and GenePept (release 165, 

2008) (Capt, 2009). Searches included epitope homology search using overlapping 

eight amino acid sequences and overall homology search utilizing BLASTP algorithm. 

The matching criteria was 100% identity match over a linear contiguous eight amino acid 

segment and 35% identity match over the full-length query sequence with a known toxin 

or allergen for epitope and overall homology searches, respectively. No significant 

similarities were found between the putative seven ORF sequences and any sequences 

from the databases. 

These findings show that the ORF analysis of the insertion present in event T303-3 did 

not identify any potential for new proteins to be expressed and moreover, putative amino 

acid sequences did not show homology to any known toxins or allergens. 

V.C. Confirmation of the Absence of Vector Backbone

Southern analysis, confirmed the absence of vector backbone in event T303-3 (Habex, 

2006). Samples of genomic DNA from event T303-3, non-transgenic control plants

(negative control), and DNA from plasmid pTDL004 (positive control) were analyzed

using seven overlapping vector backbone probes covering the vector backbone 

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sequences of plasmid pTDL004. Detailed materials and methods are provided in 

Appendix 2.B. A schematic diagram illustrating the probe strategy is shown in Figure 7. 

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Figure 7. Schematic drawing of probe strategy for vector backbone detection

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Expected and obtained hybridization fragments by probe, test material, and digest used 

are provided in Appendix 3 Table 24 (vector backbone probes) and 25 (T-DNA probe). 

Genomic DNA from event T303-3 plants and negative control plants showed no 

hybridization with any of the vector backbone probes. Hybridization occurred with the 

positive control, demonstrating the conditions were appropriate for hybridization to occur 

(Figure 8). 

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Figure 8. Event T303-3 verification of absence of vector backbone – Southern blot 

results

PT002-1 probe PT003-1 probe

PT005-1 probe PT007-1 probe

Loading Sequence All Probes

Lane 2: G. hirsutum event T303-3 – EcoRV digested

Lane 3: G. hirsutum event T303-3 – NotI digested

Lane 4: G. hirsutum wild type – NotI digested

Lane 5: G. hirsutum wild type – NotI digested + 0.1 copy pTDL004 – NotI digested

Lane 6: G. hirsutum wild type – NotI digested + 1 copy pTDL004 – NotI digested

Lane 7: G. hirsutum wild type – NotI digested + 10 copy pTDL004 – NotI digested

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Figure 8 (continued). Event T303-3 verification of absence of vector backbone – 

Southern blot results

PT008-2 probe PT009-1 probe 

PT010-1 probe PT011-1 probe

Loading Sequence All Probes

Lane 2: G. hirsutum event T303-3 – EcoRV digested

Lane 3: G. hirsutum event T303-3 – NotI digested

Lane 4: G. hirsutum wild type – NotI digested

Lane 5: G. hirsutum wild type – NotI digested + 0.1 copy pTDL004 – NotI digested

Lane 6: G. hirsutum wild type – NotI digested + 1 copy pTDL004 – NotI digested

Lane 7: G. hirsutum wild type – NotI digested + 10 copy pTDL004 – NotI digested

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V.D. Mendelian Inheritance

The Mendelian segregation of event T303-3 was analyzed, using chi-square analysis, at 

several stages of introgression of event T303-3 into conventional lines as well as in several 

inbred lines of the T0 transformant. To determine whether event T303-3 was segregating 

as expected each generation was sprayed with glufosinate ammonium herbicide to 

eliminate herbicide-susceptible plants. The results shown in Table 5 illustrate that event 

T303-3 behaves as a single dominant allele at one locus, with the expected segregation 

ratio of 3:1 and is stably integrated within and across generations. 

Table 5. Mendelian segregation of cotton event T303-3 

Generation Ratio Observed Expected ChiSquare

Tc Sd Tc Sd

T1 3:1 40 9 37 12 1.1496e

T2 3:1 23 6 22 7 0.2873e

F2a 3:1 16 6 15 5 2.5f

BC3F3b 3:1 18 6 15 9 0.3f

a Self-pollinated hemizygous plant. b Hemizygous BC3F2 plant, self-pollinated. c Tolerant to glufosinate herbicide, positive segregant d Susceptible to glufosinate herbicide, null segregant e The chi-square value at the 95% level is 3.84. To be significantly different, the calculated chi-square value 

would need to be greater than 3.84.

f The chi-square value at the 95% level is 5.99. To be significantly different, the calculated chi-square value 

would need to be greater than 5.99.

 

V.E. Conclusions

Results of the molecular characterization of event T303-3 indicate that the event contains a 

single insert containing the cry1Ab and bar gene expression cassettes. The results 

demonstrated that the inserted DNA contains the cry1Ab and bar genes and their 

respective regulatory elements in a functional order. The insert has been shown to be 

stably inherited in several different genetic backgrounds and generations using Mendelian 

segregation analysis that tracked phenotypic tolerance to glufosinate ammonium herbicide. 

Those results clearly indicate that the trait is inherited as expected for a single locus. 

Southern blot analysis also confirmed the absence of the transformation vector backbone. 

Genomic flanking sequences at the 5’ and 3’ junctions of the insertion in event T303-3 

were sequenced and found to match those of the pre-insertion locus. Bioinformatics 

analysis of ORFs did not identify any homologies with known allergen and toxin 

sequences.

With the exception of the promoter for the cry1Ab gene, event T303-3 cotton contains the 

same genetic elements as the antecedent organism. Genetic characterization 

demonstrates that event T303-3 is no more likely to become a plant pest in the 

environment than the antecedent organism, event T304-40.

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VI. CHARACTERIZATION OF THE INTRODUCED PROTEINS

Event T303-3 and the antecedent organism, event T304-40, were transformed to produce 

the same insecticidal crystal protein (ICP) Cry1Ab coded for by the cry1Ab gene isolated 

from Bacillus thuringiensis berliner and the same PAT protein expressed by the bar gene 

from Streptomyces hygroscopicus. Event T304-40 is one of the parental events in TwinLink 

Cotton (T304-40 x GHB119) and as such has been previously reviewed by USDA APHIS 

as part of the determination of non-regulated status (USDA-APHIS, 2011). 

VI.A Cry1Ab Protein

VI.A.1 History and Background

There are no changes in the history and background of the Cry1Ab protein from the 

previous petition 08-340-01p entitled “Petition for Determination of Nonregulated Status for 

Insect-Resistant and Glufosinate-Ammonium-Tolerant Cotton: TwinLinkTM Cotton (events 

T304-40 x GHB119)” (USDA, 2008). 

VI.A.2 Characterization of the Cry1Ab Protein

Petition 08-340-01p demonstrated the Cry1Ab protein produced in planta by event T304-40

was comparable to the Cry1Ab protein isolated from E. coli (USDA, 2008). For the protein

safety data provided in the petition to also be applicable to the Cry1Ab protein produced by 

event T303-3, additional analysis by SDS-PAGE was performed to demonstrate that the

Cry1Ab protein produced in planta by event T303-3 is comparable to that produced by 

event T304-40 and the E. coli-derived protein. 

Because the Cry1Ab protein in T303-3 cotton was shown to be comparable to that of event 

T304-40 (Section VI.E.), no additional information on the characterization beyond that 

presented in petition 08-340-01p is provided. 

VI.B The PAT/bar Protein

PAT protein biochemistry and mode of action are included in the peer-reviewed scientific 

journal published by Herouet et al. (2005) and in US-FDA BNF 000086 (US-FDA, 2003).

Briefly, phosphinothricin (L-PPT) and demethylphosphinothricin (DMPT) are inhibitors of 

glutamine synthetase. This inhibition results in the accumulation of toxic ammonium ions 

and a decrease in the amount of glutamine, an essential amino acid used in many anabolic

processes. The PAT enzyme is an acetyltransferase that specifically catalyzes the 

acetylation of both L-PPT and DMPT. Enzymatic properties of the PAT protein are wellcharacterized, in particular, pH and temperature dependency are well-described and 

understood. From the perspective of safety, this characterization demonstrates that 

metabolic effects of the expression of the PAT protein are limited to conferring tolerance to 

the herbicide glufosinate ammonium.

VI.C Protein Safety

Since the Cry1Ab and PAT proteins expressed in T303-3 cotton are the same as 

expressed in T304-40 cotton, no additional safety information is available that was not 

presented in petition 08-340-01p. In summary, in order to assess any potential adverse 

effects to humans or animals resulting from environmental release of the crops expressing

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the Cry1Ab and PAT proteins, Bayer CropScience (BCS) has conducted a detailed safety 

evaluation based on Codex Alimentarius Commission (Codex Alimentarius, 2003). As a 

basis, BCS performed a series of safety studies with these proteins, including homology 

searches of the amino acid sequences with comparison to all known allergens and toxins 

from large public databases, an in vitro digestibility assay of the proteins, and an acute 

toxicity test in the mouse. Moreover, publicly available review documents issued by 

regulatory authorities, indicating that similar protein family members are safe, have been 

used for supporting this safety assessment. The results of studies conducted by BCS are 

consistent with the published information, confirming that the crops containing these 

proteins can be safely used as food or feed.

Lack of allergenic potential

• The Cry1Ab and PAT proteins have no amino acid sequence similarity to known

allergens, as demonstrated by overall amino acid and epitope homology searches;

• As expected, the Cry1Ab and PAT proteins only have similarities with other Cry or

PAT proteins;

• The Cry1Ab and PAT proteins do not share epitopes with known allergens;

• The Cry1Ab and PAT proteins are not glycosylated;

• The Cry1Ab and PAT proteins are not heat stable;

• The Cry1Ab and PAT proteins are degraded by human simulated gastric and

intestinal fluids. This minimizes the likelihood that these proteins could survive in 

the human digestive tract and be absorbed.

Lack of toxic potential

• The Cry1Ab and PAT proteins have no amino acid sequence similarity to known

allergens, as demonstrated by overall amino acid homology searches;

• The Cry1Ab and PAT proteins are degraded by human simulated gastric and

intestinal fluids. This minimizes the likelihood that these proteins could survive in 

the human digestive tract and be absorbed;

• There were no mortalities, clinical signs or treatment-related effects in female OF1 

mice after an acute oral administration by gavage of the Cry1Ab, Cry2Ae or PAT 

proteins at 2,000 mg protein/kg body weight.

In conclusion, it is considered that the cry1Ab and bar genes as well as the Cry1Ab and 

PAT proteins are not toxic to mammals and do not possess any of the characteristics 

associated with food allergens. Therefore, no effects on animal and human health are to be 

expected by consumption of the cry1Ab or bar genes and the Cry1Ab or PAT proteins.

VI.D. Expression of Introduced Proteins

The content of the Cry1Ab and PAT proteins in ginned cottonseed (also referred to as 

fuzzy seed) of event T303-3 was determined by enzyme-linked immunosorbent assays 

(ELISA) specific to each protein (Robinson, 2008). Detailed methods for analysis of 

expression levels of Cry1Ab in raw agricultural commodity are provided in Appendix 2.C. 

Since fuzzy seed is not delinted, it could not be ground into a homogeneous material. A 

procedure was developed previously to effectively remove the lint coat from the kernel, 

creating two fractions (kernel and lint coat). These two fractions were analyzed separately 

for Cry1Ab and PAT content and then added together to re-create the amount in the 

original fuzzy seed. Seed was collected from event T303-3 plants grown in six field trials in 

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the U.S. One unsprayed event T303-3 sample and three event T303-3 samples sprayed 

with glufosinate ammonium from each of the sites were analyzed. Unsprayed, nontransgenic seed (control) from each of the trial sites was also analyzed.

On a dry weight basis, the average Cry1Ab protein content from all test sites of cotton 

fuzzy seed samples from unsprayed event T303-3 plants ranged from 7.14 μg/g to 17.9 

μg/g (overall average value = 11.7 ± 3.74 μg/g) and from 10.9 μg/g to 19.1 μg/g in fuzzy 

seed samples from sprayed event T303-3 plants (overall average value = 14.3 ± 3.50 μg/g)

(Table 6). Cry1Ab protein constitutes 0.005073% of the total crude protein in unsprayed 

transgenic fuzzy seed and 0.005862% in sprayed transgenic fuzzy seed. No Cry1Ab 

protein was detected in the non-transgenic control fuzzy seed samples. As a comparison, 

the Cry1Ab content of event T304-40 in unsprayed fuzzy seed was 1.29 + 0.52 μg/g d.w. 

(0.000561% of total crude protein) and 1.54 + 0.318 μg/g d.w. (0.000561% of total crude 

protein). These differences in the amount of Cry1Ab protein does not affect the efficacy

displayed toward the target pest organisms and the Cry1Ab protein represents a small 

component of the total protein in events T303-3 and T304-40.

Table 6. Cry1Ab protein levels in event T303-3 fuzzy seed

Sample Average Cry1Ab Content

(μg/g Sample)

Average Cry1Ab Content

(as % of total crude protein)

No Glufosinate Glufosinate Sprayed No Glufosinate Glufosinate Sprayed

Fuzzy Seed 17.9 17.3 0.007668 0.007442

11.4 15.4 0.005758 0.006873

7.14 10.9 0.003004 0.004631

11.3 11.8 0.004450 0.004436

9.06 11.3 0.003901 0.004255

13.7 19.1 0.005659 0.007537

Range in 

Values 7.14 – 17.9 10.9 – 19.1 0.003004 –0.007668

0.004255 –

0.007537

Average ± SD 11.7 ± 3.74 14.3 ± 3.50 0.005073 ±

0.001648

0.005862 ±

0.001578

On a dry weight basis, the average PAT protein content from all test sites of transgenic 

cotton ranged from 56.5 μg/g to 130 μg/g d.w. in unsprayed fuzzy seed (overall average 

value = 102 + 26.9 μg/g) and from 89.3 μg/g to 142 μg/g in sprayed fuzzy seed (overall 

average value = 118 + 20.7 μg/g) (Table 7). PAT protein constitutes 0.043618% of the 

total crude protein in unsprayed transgenic fuzzy seed and 0.048027% of the total crude 

protein in sprayed transgenic fuzzy seed. No PAT/bar protein was detected in the nontransgenic unsprayed fuzzy seed. As a comparison, the PAT/bar protein of event T304-40 

was 163 + 32 μg/g (0.0728% of total crude protein) in unsprayed transgenic fuzzy seed 

and 163 + 27 μg/g (0.0736% of total crude protein) in sprayed transgenic fuzzy seed. 

These differences in the amount of the PAT/bar protein do not affect the level of tolerance 

to the herbicide between the two events and PAT protein represents a small component of 

the total protein content of the two events.

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Table 7. PAT/bar protein levels in event T303-3 fuzzy seed 

Sample Average PAT/bar Content

(μg/g Sample) ± SD

Average PAT/bar Content

(as % of crude protein) ± SD

No Glufosinate Glufosinate Sprayed No Glufosinate Glufosinate Sprayed

Fuzzy Seed 113 115 0.048430 0.049645

82.7 101 0.041649 0.045095

56.5 89.3 0.023766 0.038042

114 142 0.044902 0.053390

130 125 0.055968 0.047537

113 138 0.046993 0.054451

Range in 

Values 56.5 – 130 89.3 – 142 0.023766 –0.055968

0.038042 –

0.054451

Average ± SD 102 ± 26.9 118 ± 20.7 0.043618 

±0.010832

0.048027 

±0.006020

VI.E Comparison of Cry1Ab Proteins from Events T303-3 and T304-40

Safety assessment studies, such as the acute toxicity study, require large amounts of the 

target protein. The expression of the Cry1Ab protein in event T303-3 cotton tissues is 

relatively low and thus, purification of sufficient quantities of the protein for safety 

assessment studies from cotton tissue is not feasible. Thus, Cry1Ab protein was produced 

using E. coli. The protein produced by the bacterium was engineered to match the amino 

acid sequence of the Cry1Ab protein produced in planta. A full protein characterization 

study (SDS-PAGE, western analysis, glycol-staining, LC/MS, N-terminal sequencing) has 

been conducted on the Cry1Ab protein, expressed by event T304-40, in the combined 

event product TwinLink (T304-40 x GHB119) (USDA, 2008). Therefore, a SDS-PAGE 

analysis was conducted to compare the Cry1Ab protein isolated from event T303-3 cotton 

plants to the Cry1Ab protein isolated from event T304-40 cotton plants and the microbiallyproduced Cry1Ab from E. coli (Haas, 2009). A summary of the methods is provided in 

Appendix 2.D. 

The Cry1Ab protein isolated from event T303-3, event T304-40 and E. coli was analyzed 

by SDS-PAGE to compare their respective molecular weight. Figure 9 shows the 

Coomassie stained gel. The Cry1Ab from events T303-3 and T304-40 have equivalent 

measured mobilities of 25 mm. The mobility of the trypsin digested Cry1Ab from E. coli

(26mm) is greater than that of the plant-derived Cry1Ab proteins due to the loss of 28 

amino acids which were cleaved during the production of the protein.

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Figure 9. Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein isolated from events T303-3 and T304-40

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The Coomassie stained gel in Figure 10 shows a comparison of the electrophoretic mobility

of trypsin digested Cry1Ab protein from E. coli and a thrombin-engineered Cry1Ab protein 

from E. coli to the Cry1Ab isolated from event T304-40 cotton leaves. The thrombinengineered Cry1Ab protein is representative of the plant form of Cry1Ab expressed in 

events T303-3 and T304-40 cotton and is converted to the active form when ingested by 

the target insect. The measured mobility of this protein and the Cry1Ab protein from event 

T304-40 cotton leaves was 32mm. The measured mobility of the trypsin-digested Cry1Ab 

was 34mm. Plotting of the electrophoretic mobility vs. the respective molecular weight

(MW) yielded the approximate molecular weight. For the Cry1Ab protein from the T304-40 

cotton leaves and the thrombin protein standard, the approximate MW was 68.2 kDa. The 

approximate MW for the trypsin-digested protein was 62.6 kDa.

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Figure 10. Comparison of trypsin-digested and thrombin-engineered Cry1Ab from E. coli to Cry1Ab isolated from event T304-40

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In summary, the SDS-PAGE results indicate that the E. coli-produced Cry1Ab proteins are 

comparable to the Cry1Ab proteins isolated from events T303-3 and T304-40. Therefore, 

protein characterization and safety data for the Cry1Ab produced by event T304-40 

provided in petition 08-340-01p adequately addresses the same criteria for event T303-3.

 

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VII. AGRONOMIC AND PHENOTYPIC EVALUATION

Agronomic and phenotypic characteristics of event T303-3 were evaluated in field trials in 

multiple locations in typical cotton growing regions of the US in 2005 and 2006. Field trials 

were conducted according to common agricultural practices for a given growing region. 

In addition, all field trials of T303-3 conducted from 2001 to 2006 for increasing research 

seed as well as agronomic and phenotypic evaluation were observed for unusual or 

unexpected characteristics in regards to susceptibility to plant disease, weediness 

potential, effect on non-target organisms, beneficial insects, and the overall environment

(Appendix 1). 

VII.A History of Field Activities

Field trials for characterization of agronomic and plant product quality (i.e., fiber quality 

parameters) were conducted in 2005 under acknowledged notification 05-040-06n. In 

2006 field trials were conducted under acknowledged notification 06-047-02n to assess 

agronomics, plant product quality, and phenotypic characteristics (i.e., efficacy). A 

summary of field trial locations for the assessment of agronomics, plant product quality, 

and phenotypic characteristics is provided in Table 8. Locations for agronomic and 

phenotypic evaluation were representative of major portions of the US Cotton growing 

regions including the Texas High Plains and Gulf Coast, and the Louisiana Delta region. 

Table 8. Field trial locations for evaluation of agronomic and phenotypic 

characteristics

Notification 

Number Year Number of Locations Locations

05-040-06n 2005 3 

Gaines Co., TX

Uvalde Co., TX

Wharton Co., TX 

06-047-02n 2006 5 

Bossier Co., LA

Gaines Co., TX

Lubbock Co., TX

Wharton Co., TX (2)a 

 a Two sites at this location

Field trials conducted in 2005 for agronomic and plant product quality assessment included 

homozygous event T303-3 of Coker 315 (T4 generation) compared with wild type Coker 

315. 

In 2006, comparisons for agronomics and plant product quality were assessed in four field 

trials. Phenotype (i.e., efficacy) parameters were assessed in up to five field trials;

however fewer field trials were utilized for the assessment of certain parameters. Field

trials in 2006 utilized homozygous event T303-3 in the Coker 315 background (T5

generation) and wild type Coker 315 as the comparator. In each of these trials, entries of

homozygous event T303-3 and the comparator were infested or not infested with larva of 

Helicoverpa zea Boddie (cotton bollworm). Plots which were not artificially infested were 

sprayed with conventional insecticides according to university extension recommendations 

if natural infestations were observed. 

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Similar field trials were used for assessment of the antecedent organism, event T304-40, in 

2007 and 2008. 

VII.B Agronomic and Phenotypic Characteristics Evaluated

A summary and descriptions of agronomic, yield and plant product quality, and phenotypic

characteristics evaluated in 2005 and 2006 field trials are provided in Table 9, 10, and 11. 

Table 9. Agronomic characteristics evaluated in field trials

Parameter Description

Emergence Evaluation of the germination rates and plant population

Height Average plant height from cotyledonary node to terminal, 

expressed in inches

Total number of nodes Number of reproductive nodes present on the main stem of 

the plant

Height to node ratio (HNR) Plant height divided by total number of nodes

Table 10. Yield and fiber quality characteristics evaluated in field trials

Parameter Description

Yield Productivity expressed as pounds of lint produced per acre

Gin turnout Lint weight divided by seed cotton weight, expressed as a 

percentage

Seeds per boll Average number of seeds per boll

Seed index Average weight in grams of 100 seed, an indication of seed 

size and maturity.

Fiber length Average length of the longer one-half of cotton fibers

Fiber strength

The force in grams required to break a bundle of fibers one 

tex unit in size (1 tex = weight in grams of 1,000 meters of 

fiber)

Fiber micronaire A measure of fiber fineness and maturity as indicated by 

specific surface area

Fiber uniformity % Ratio between the mean length and upper half mean length 

of the fibers expressed as a percentage

Table 11. Phenotypic (efficacy) characteristics evaluated in field trials

Parameter Description

Flowers- % damaged A measure of the percent open flowers damaged by insects

Flowers- living larvae A count of living insect larva present in 25 flowers

Squares- % damaged A measure of the percent immature flowers (squares) 

damaged by insects 

Squares- living larvae A count of living insect larva present in 25 squares

Bolls- % damaged A measure of the percentage of fruit (bolls) damaged by 

insects

Bolls- living larvae A count of living insect larva present in 25 bolls

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Agronomic, plant product quality, and phenotype parameters were not assessed at all 

locations each year. Table 12 provides a matrix of which parameters were assessed at 

each field trial location.

Table 12. Parameters assessed by year and location

Location

County/ST Year

EmergenceHeightTotal nodesHNRYieldGin turnoutSeeds per bollSeed indexFiber lengthFiber strength Fiber micronaireFiber uniformityFlowers-% damagedFlowersliving larvaeSquares-% damagedSquaresliving larva

e

Bolls - % damagedBolls -living larva

e

Gaines/TX ‘05 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Uvalde/TX ‘05 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Wharton/TX ‘05 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Bossier/LA ‘06 ■ ■ ■ ■

Gaines/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Lubbock/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Wharton/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

Wharton/TX ‘06 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

VII.B.1 Agronomic and Plant Product Characteristics

In 2005, some small differences were observed in agronomic characteristics between 

homozygous event T303-3 cotton and the comparator. In most cases, event T303-3 cotton 

was within the ranges observed for TwinLink Cotton (the product of the breeding cross of 

event GHB119 and event T304-40, the antecedent organism). A summary of agronomic 

data from 2005 field trials across locations is presented in Table 13 along with range of

means for TwinLink Cotton from 2007 and 2008 field trials for the same parameters. 

Table 13. Summary of agronomic data from 2005 field trials

Parameter

Coker 315

WT

Mean

Coker 315

T303-3 

Mean

Standard 

Deviation

TwinLink

Range of Meansa 

Emergence

plants / ft

(3)b

0.44 0.23 0.098 1.36 – 3.57

Height

inches

(3)

38.81 41.16 2.210 30.33 – 38.17

Total Nodes

(3) 22.34 24.67 0.712 16.95 – 23.00

HNR

(3) 1.74 1.67 0.040 1.62 – 3.74

aRange of means for TwinLink Cotton (GHB119 x T304-40) from petition 08-340-01p bNumber of field trials where parameter was measured

WT = Wild type

HNR = Height to node ratio 

Generally, event T303-3 Coker 315 and wild type Coker 315 were comparable for most 

parameters and means were within one standard deviation of each other. Further, 

agronomic parameters for event T303-3 fell within the range of values observed for 

TwinLink Cotton, or at most approximately one standard deviation outside that of TwinLink 

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Cotton. Comparisons of TwinLink Cotton to event T303-3 should be made with the 

consideration that the data does not come from the same field trials or the same year and 

therefore variation due to weather and location is expected. 

In 2005, emergence was particularly low for both wild type Coker 315 and event T303-3. 

This was due to poor seed quality as a result of the delinting process for preparing planting 

seed. The process for preparing planting seed was improved upon for the 2006 field trials 

as evidenced by markedly improved emergence for both wild type Coker 315 and event 

T303-3 cotton. In 2006 field trials, all agronomic parameters evaluated were in line with 

the observed values for TwinLink Cotton from 2007 and 2008 field trials (Table 14). 

Table 14. Summary of agronomic data from 2006 field trials

Parameter Coker 315

WT (I)

Coker 315

WT

Coker 315

T303-3 (I)

Coker 315

T303-3

Standard 

Deviation

TwinLink

Range of Meansa 

Emergence

plants / ft 

(4)b

3.53 3.65 3.07 3.49 0.473 1.36 – 3.57

Height

inches

(4)

38.46 37.43 38.66 38.02 1.196 30.33 – 38.17

Total Nodes

(4) 20.67 19.87 21.33 20.88 0.518 16.95 – 23.00

HNR

(4) 1.84 1.87 1.80 1.82 0.077 1.62 – 3.74

aRange of means for TwinLink Cotton (GHB119 x T304-40) from petition 08-340-01p Appendix 4 bNumber of field trials where parameter was measured

WT = Wild type

(I) = Infested with H. zea

HNR = Height to node ratio 

Plant product characteristics were generally comparable among event T303-3 and wild 

type Coker 315 in 2005 field trials (Table 15). All plant product characteristics of event 

T303-3, with the exception of micronaire, were within the range of means observed for 

TwinLink Cotton in 2007 and 2008 field trials. In the case of micronaire, event T303-3 was 

within one standard deviation of that observed for TwinLink Cotton. Seed per boll was not 

evaluated in TwinLink Cotton field trials and therefore is not presented in Table 15. 

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Table 15. Summary of plant product characteristics from 2005 field trials

Parameter

Coker 315

WT

Mean

Coker 315

T303-3 

Mean 

Standard 

Deviation

TwinLink

Range of Meansa

Yield 

lb / A

(3)b

1180.56 749.22 68.657 608.00 – 1434.00

Gin Turnout

% Lint

(3)

39.74 38.91 0.619 35.97 – 45.40

Seed per Boll

(3) 11.06 12.00 0.534 - - - 

Fiber Length

1/32nd inch (3) 1.16 1.25 0.001 1.11 – 1.30

Fiber Strength 

g / TEX

(3)

29.20 30.98 0.869 30.05 – 34.77

Micronaire

(3) 4.09 3.69 0.249 3.77 – 5.25

Fiber 

Uniformity

%

(3)

83.06 85.60 0.403 81.75 – 87.87

aRange of means for TwinLink Cotton (GHB119 x T304-40) from petition 08-340-01p bNumber of field trials where parameter was measured

WT = Wild type

HNR = Height to node ratio 

Again in 2006 field trials, plant product characteristics were generally comparable between 

wild type Coker 315 and event T303-3 in both the infested and non-infested treatments 

(Table 16). Means for event T303-3 from either treatment were generally within the ranges 

of that observed for TwinLink Cotton. Micronaire, fiber strength and yield were within one 

standard deviation of that observed for TwinLink Cotton. Again seed per boll and seed 

index were not evaluated in TwinLink Cotton field trials. 

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Table 16. Summary of plant product characteristics from 2006 field trials

Parameter Coker 315

WT (I)

Coker 315

WT

Coker 315

T303-3 (I)

Coker 315

T303-3

Standard 

Deviation

TwinLink

Range of Meansa 

Yield 

lb / A

(4)b 

756.19 942.08 584.36 613.00 73.404 608.00 – 1434.00

Gin Turnout

% Lint

(4)

38.03 38.29 37.62 37.37 1.092 35.97 – 45.40

Seed per Boll

(4) 30.90 32.70 22.42 22.71 1.202 - - - 

Seed Index

g / 100 seed

(4)

7.99 7.69 9.05 8.51 0.749 - - - 

Fiber Length

1/32nd inch (4) 1.17 1.16 1.24 1.25 0.020 1.11 – 1.30

Fiber Strength 

g / TEX

(4)

28.65 28.80 29.33 28.87 0.745 30.05 – 34.77

Micronaire

(4) 4.27 4.06 3.56 3.46 0.252 3.77 – 5.25

Fiber 

Uniformity

%

(4)

82.92 82.37 82.84 83.12 0.405 81.75 – 87.87

aLow and high mean for TwinLink Cotton (GHB119 x T304-40) from petition 08-340-01p Appendix 4 bNumber of field trials where parameter was measured

WT = Wild type

(I) = Infested with H. zea

Event T303-3, when compared with wild type Coker 315, consistently had lower yields in 

both 2005 and 2006 field trials. This in part led to the decision to move forward with the 

antecedent organism, event T304-40, as the “Cry1Ab component” in the TwinLink Cotton 

breeding stack. Aside from yield, event T303-3 was comparable to wild type Coker 315 

and TwinLink cotton in all other agronomic and plant product characteristics. Though 

event T303-3 demonstrated lower yield potential, this does not in itself present any greater 

risk of event T303-3 becoming a plant pest. 

VII.B.2 Assessment of Phenotype by Efficacy Against Insect Pests

The efficacy assessments conducted in 2006 field trials showed that the phenotype of 

event T303-3 was performing as expected and that the Cry1Ab protein expressed in event 

T303-3 was effective in protecting the plant from cotton bollworm. 

A summary of efficacy data is presented in Table 17. As expected, event T303-3 in either 

the infested or non-infested treatments had lower damage ratings on squares, flowers, or 

bolls compared to the wild type Coker 315, infested or not infested with cotton bollworm. 

The same is true in terms of living larvae of cotton bollworm found on squares, flowers, or 

bolls. For all assessments made, event T303-3 plots that were infested or not infested 

were similar to each other. These results confirm that the Cry1Ab protein expressed by 

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event T303-3 effectively controls the target insect pest it was challenged with in these field 

trials and the phenotype of event T303-3 is comparable to the antecedent organism. 

Table 17. Summary of phenotypic characteristics by efficacy against target pests

Parameter Coker 315

WT (I)

Coker 315

WT

Coker 315

T303-3 (I)

Coker 315

T303-3

Standard 

Deviation

Flowers 

% Damaged

(3)a

8.8 3.8 1.9 1.5 1.984

Flowers

Living Larvae

(3)

1.73 1.50 0.40 0.20 0.666

Squares

% Damaged

(5)

19.63 10.86 2.20 1.14 5.716

Squares

Living Larvae

(4)

3.27 1.77 0.28 0.14 1.708

Bolls

% Damaged

(5)

5.56 1.97 1.72 0.50 1.477

Bolls

Living Larvae

(4)

0.34 0.23 0.14 0.06 0.171

aNumber of field trials where parameter was measured

WT = Wild type

(I) = Infested with H. zea

VII.C Disease and Pest Characteristics

Personnel conducting the field trials also visually monitored plant disease and pest 

resistance characteristics of event T303-3 cotton and non-transgenic controls. There were 

no differences reported in severity of disease symptoms or insect damage (other than the 

targeted insects susceptible to the Cry1Ab protein) between the transgenic plants and the 

non-transgenic cotton plants. For additional information please see the field trial 

termination reports provided in Appendix 1. 

VII.D Composition Analysis

To conduct compositional analysis of event T303-3 cotton, six field trials were conducted in 

Georgia, Arkansas, Mississippi and Texas, all important cotton-growing regions of the 

southern U.S. The plants in the trials were grown under conditions typical of cotton 

production practices. At each site, there were six transgenic plots and three nontransgenic plots. Three of the transgenic plots at each site were sprayed twice with a tank 

mix of glufosinate ammonium herbicide at 0.52 lb a.i./A and ammonium sulfate at 3.0 lbs/A 

(target application rate). The other three transgenic plots at each site were left unsprayed. 

Nine samples of fuzzy seed (ginned cottonseed) were collected from each of the six field 

trials (Haas, 2007). There were a total of 18 samples from each of three groups: nontransgenic Coker 315 cotton, T303-3 cotton that was unsprayed and T303-3 cotton that 

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was sprayed with glufosinate herbicide. Analyses of the seed samples were carried out at 

Eurofins Scientific Des Moines, IA (Table 18). For list of the analytical methods used refer 

to Table 21 in Appendix 2.E. 

Table 18. Analyses performed on cottonseed of event T303-3 and its non-transgenic 

counterpart

VII.D.1 Proximate Analysis

 

Table 19 shows the results of the proximate analysis of event T303-3 cottonseed. These 

results are comparable to the proximate analysis of TwinLink (T304-40 x GHB119) as 

provided in petition 08-340-01p (USDA, 2008). 

Table 19. Mean proximate composition in cottonseed of event T303-3 

Parameter

NonTransgenicTransgenic

Not sprayed

Transgenic

Sprayed Reference 

ranges a

Mean ± SD Mean ± SD Mean ± SD

Moisture %fw 8.64 ± 1.77 9.42 ± 1.34 9.71 ± 1.91 4.0 – 15.9

Protein %dm 23.01 ± 2.06 24.05 ± 1.70 24.63 ± 1.83 11.7 – 34.2

Fat %dm 17.38 ± 2.90 19.05 ± 1.37 19.13 ± 1.46 11.8 – 36.3

Ash %dm 4.26 ± 0.56 4.53 ± 0.45 4.65 ± 0.57 3.2 – 5.0

Total 

Carbohydrates %dm b 55.35 ± 3.29 52.37 ± 2.08 51.60 ± 2.47 36.4 – 74.4

ADF %dm 40.58 ± 4.56 39.48 ± 4.20 36.80 ± 3.46 29.0 – 66.9

NDF %dm 50.02 ± 5.90 48.85 ± 4.03 45.80 ± 4.47 38.1 – 71.4

Data represent an average of three replicate samples at six field test sites.

a Reference ranges compiled from OECD (2004), ILSI (2007), USCA (1982), Calhoun et al. (1995), Bertrand et 

al. (2005), Amann (1999), Lundquist (1995), Berberich et al. (1996) and Nida et al. (1996). b Total carbohydrates calculated as 100% - (protein %dm + fat %dm + ash %dm)

Additional composition data on amino acids, fatty acids, minerals and vitamins is provided 

in Appendix 3.C. Literature values for the individual analyses of cottonseed are also 

presented for reference. Values for event T303-3, in either the sprayed or unsprayed 

treatment, were comparable with those the non-transgenic counterpart and within the 

reference ranges from the literature.

VII.D.2 Anti-Nutrient Analysis

Fuzzy seed of event T303-3 was also analyzed for the content of anti-nutrients of cotton—

gossypol and phytic acid (Haas, 2007). The results are presented in Table 20. The values 

for gossypol and phytic acid were within the reported literature ranges and were similar for 

the non-transgenic, the transgenic unsprayed and transgenic sprayed plants and 

comparable to that of TwinLink Cotton (USDA, 2008).

Matrix Analyses Performed 

Cottonseed (fuzzy seed) Proximates: moisture, ash, fat, protein, including 

carbohydrate calculation; acid detergent fiber; neutral 

detergent fiber, 

Antinutrients: gossypol (free,”-“, “+” and total); phytic 

acid

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Table 20. Mean anti-nutrient composition in cottonseed of event T303-3 

Parameter

Based on dry matter

Reference 

rangesa

NonTransgenicTransgenic

Not sprayed

Transgenic

Sprayed

Mean ± SD Mean ± SD Mean ± SD

Free gossypol % 0.53 ± 0.12 0.56 ± 0.06 0.53 ± 0.06 0.23 – 1.40

(−)Gossypol % 0.32 ± 0.07 0.32 ± 0.05 0.32 ± 0.04 0.18 – 0.77b

(+)Gossypol % 0.41 ± 0.08 0.44 ± 0.04 0.43 ± 0.05 0.28 – 1.22b

Total gossypol % 0.73 ± 0.15 0.76 ± 0.08 0.75 ± 0.08 0.46 - 1.99

Phytic acid % 1.63 ± 0.29 1.65 ± 0.25 1.70 ± 0.28 0.85 - 2.57

Data represent an average of three replicate samples at six field test sites. 

aReference ranges compiled from OECD (2004), ILSI (2007), Calhoun et al. (1995), Berberich et al. (1996), 

Nida et al. (1996)., Phelps et al. (1965), and Wozenski and Woodburn (1975) bThe proportion of (-)gossypol and (+) gossypol in whole, fuzzy cottonseed was reported as 38.8% and 61.2% 

of total gossypol (Calhoun et al., 1995); on the basis of the range for total gossypol of 0.46 – 1.99 % dm this 

equals to 0.18 – 0.77% dm for (-) gossypol and 0.28 – 1.22% dm for (+) gossypol.

VII.E. Conclusion

Overall, these evaluations indicate that event T303-3 does not show any unexpected 

changes in plant morphology as compared to conventional cotton. Any statistically 

significant differences observed are unrelated to the introduced trait. The agronomic 

performance data indicate no biologically meaningful differences between event T303-3 

and the non-transgenic counterpart nor the antecedent organism, event T304-40. No 

differences between the event T303-3 and the non-transgenic control were observed for 

disease occurrence or severity and response to insect pressure, with the exception of the 

target pests.

Samples of fuzzy seed of event T303-3 were analyzed for their proximate, amino acids, 

fatty acids, minerals, vitamins and anti-nutrient composition and compared to a nontransgenic control. Published data ranges were provided as additional points of reference. 

Plant material was collected from six locations and analyzed using standards methods. 

The ranges and standard deviations overlapped for event T303-3 and the non-transgenic 

control and fell within the published data ranges.

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VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTICES

VIII.A. Potential for Gene Transfer

Only two wild Gossypium species are present in the US: G. thurberi Todaro found in 

mountain regions of Arizona at altitudes of 2500 to 5000 feet and G. tomentosum which is 

found in Hawaii. Only G. tomentosum is capable of crossing with domesticated cotton that 

will produce fertile offspring. There is no expected selective advantage conferred by the 

introduced cry1Ab and bar genes in event T303-3 if that cross would occur.

Vertical Gene Flow: 

Cotton pollination

Gossypium hirsutum is considered to be a self-pollinating crop. Cotton pollen is heavy and 

sticky thus cross pollination by wind is unlikely. Cotton can, however, be pollinated by 

insects. Honeybees (Apis mellifera) and bumblebees (Bombus spp.) are the primary insect 

pollinators. McGregor (1976) traced the movement of pollen from a cotton field surrounded 

by a large number of honeybee colonies. Movement of the pollen was traced by means of 

fluorescent particles. McGregor found that at 150 to 200 feet away from the source plant, 

only 1.6 percent showed the presence of the fluorescent particles. By comparison, the

isolation distances for Foundation, Registered and Certified seeds in 7 CFR Part 201 are 

1320, 1320 and 660 feet, respectively. The trend for cross pollination to decrease as the 

distance from the source increased has been established by several research groups over 

the years. (Kareiva et al., 1994; Van Deynze et al., 2005).

Outcrossing potential to wild/weedy relatives

The potential for outcrossing can be defined as the ability of gene escape to wild cotton

relatives. Previously, the USDA stated in the environmental assessment document of

LLCotton25 cotton that “gene flow from genetically engineered cotton into wild cotton 

relatives are not likely, and if it occurs, would not lead to increased weediness” (USDA, 

2003). G. tomentosum, found only in Hawaii, is the only species capable of crossing with 

domesticated cotton that will produce fertile offspring. Outcrossing to G. tomentosum is 

unlikely as there is no cotton production in Hawaii other than winter nursery breeding 

activities where isolation practices are employed, and therefore the potential for gene flow 

to these wild relatives is low. There are other wild relatives known to exist in Southern 

Florida and Puerto Rico that are capable of crossing with cultivated cotton. However, these 

wild relatives are found hundreds of miles from where cotton production occurs.

Outcrossing potential to feral or cultivated cotton

No feral cotton populations (domesticated plants capable of surviving outside of cultivation) 

of G. barbadense have been found in the US Cotton production area. Seed production 

fields (production of planting seed) are required to be isolated from other cotton fields to 

prevent cross pollination. Therefore if any cross pollination were to occur to either G. 

barbadense or G. hirsutum it would be from a lint production field where seed is crushed 

and not propagated.

Potential for Horizontal Gene Flow:

Bayer CropScience is not aware of any reports of incidents of naturally occurring transgene 

movement from transgenic crops to sexually incompatible species.

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VIII.B Weediness Potential

In the United States, cotton (G. hirsutum) is not a weed pest and has no sexually 

compatible weedy relatives except perhaps G. tomentosum in Hawaii where there is no 

commercial cotton production. A number of references confirm the lack of weediness of 

cotton such as Crockett (1977), Holm et al. (1979), and Muenscher (1980). The USDA has 

previously determined that “cotton is not considered to be a serious, principal or common 

weed pest in the US” (USDA, 2003). Previous findings by the USDA of similar herbicidetolerant cotton during environmental assessment expected no change in weediness 

potential, and an example is glufosinate ammonium tolerant cotton (LibertyLink), 

commercially sold today. The largest concern is that of volunteer plants that could become 

weedy in subsequent years. Volunteers are also limited by the geography in which they

may exist as cotton does not survive as a perennial where freezing temperatures are 

reached during the winter. Volunteers can easily be controlled by crop rotation, tillage 

and/or pre- or post-emergence herbicides. For example, glufosinate ammonium tolerant 

cotton volunteers could easily be controlled by using the herbicide glyphosate.

There is limited probability that event T303-3 cotton or any Gossypium species containing 

event T303-3 cotton would become a weed problem. The data presented in this petition

did not indicate any significant differences in phenotypic or plant morphological

characteristics between event T303-3 cotton and the conventional non-transgenic 

comparator line that would impact plant pest or noxious weed potential. Based on this data 

there is no evidence to suggest that T303-3 cotton has a higher likelihood to become a 

plant pest than conventional cotton or event T304-40 cotton which has been granted nonregulated status as part of the deregulation of TwinLink Cotton on September 23, 2011 

(USDA, 2011). There were no instances in which volunteer monitoring after harvest

revealed any differences in survival or persistence relative to other cotton varieties.

VIII.C Effects on Non-Target Organisms

An assessment of the risk to non-target species has been performed for the Cry1Ab protein 

expressed in T304-40 cotton. Since the Cry1Ab protein in T303-3 cotton is the same 

protein, the assessment for T304-40 informs the assessment of T303-3. The toxicity of the 

Cry1Ab protein expressed in T303-3 cotton is expected to be similar. Testing with 

microbially-derived Cry1Ab protein at levels greatly exceeding the expressions levels found 

in Cry1Ab transgenic plants resulted in no effects on several beneficial species (USDA, 

2008). Therefore, the data presented in the previous petition adequately addresses toxicity 

to humans and non-target organisms for event T303-3 plants.

VIII.D Effects on Endangered Species

The responsibility for endangered species falls to the US Fish & Wildlife Services (FWS) 

under the Endangered Species Act (ESA) (16 USC §1531). Section 6 of the ESA requires 

federal agencies who conduct activities which may affect listed species to consult with the 

FWS to ensure that listed species are protected should there be a potential impact. 

It is not anticipated that the use of event T303-3 cotton will impact any currently listed 

species of concern. Species of concern that may inhabit areas close to commercial cotton 

operations would not be additionally impacted by the use of T303-3 cotton. Commercial 

agriculture routinely disturbs the ground in which crops are currently planted. As a result, 

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Page 50 of 105
perennial vegetative species would not grow in these areas. Additionally, because 

horizontal gene flow to sexually incompatible species is not an issue, there is negligible 

potential for exposure to the transgene contained in event T303-3 cotton through sexual 

reproduction.

Cry1Ab protein is highly specific to lepidoptera, so the only organisms which might be

considered to be ‘at risk’ from T303-3 cotton would be endangered lepidopteran insects.

While there are endangered lepidopteran species in cotton growing counties (e.g. Kern 

Primrose Sphinx moth, Saint Francis' Satyr butterfly) the larvae are highly unlikely to be 

exposed to Cry1Ab protein because their habitats do not overlap with cotton fields and their 

larvae do not feed on cotton. The amount of pollen that would drift from these cotton plants 

onto plants fed upon by endangered or threatened species would be very small. It is also 

therefore highly unlikely that event T303-3 cotton could outcross to any wild or weedy 

relatives of cotton.

For these reasons, it is not believed that the use of T303-3 cotton in commercial cotton

production will adversely impact endangered species of concern.

VIII.E Indirect Effects on Other Agricultural Products

T303-3 cotton is an alternative cotton product that contains insect-resistant and herbicidetolerant traits. Current agricultural practices already in use for these types of products are 

not expected to change with the introduction of event T303-3 cotton. Adoption and use of 

genetically modified cotton can provide positive impacts on agricultural practices. These 

positive impacts have been detailed in a study by Brookes and Barfoot (2008) and include:

Herbicide-tolerant crops

• Increased management flexibility that comes from a combination of the ease of use 

associated with broad-spectrum, post-emergent herbicides;

• Compared to conventional crops, where post-emergent herbicide application may result 

in ‘knock-back’ (some risk of crop damage from the herbicide); this problem is less likely to 

occur in herbicide-tolerant crops;

• Facilitation of adoption of no/reduced tillage practices with resultant savings in time and

equipment usage;

• Improved weed control has reduced harvesting costs – cleaner crops have resulted in 

reduced times for harvesting;

• Elimination of potential damage caused by soil-incorporated residual herbicides in followon crops.

Insect-resistant crops

• Production risk management/insurance purposes – taking away the worry of significant 

pest damage occurring;

• A ‘convenience’ benefit (less time spent on crop walking and/or applying insecticides);

• Savings in energy use – mainly associated with less spraying;

• Savings in machinery use (for spraying and possibly reduced harvesting times);

• Improved health and safety for farmers and farm workers (from reduced handling and use 

of insecticides);

Genetically modified herbicide-tolerant cotton was first grown commercially in the US in 

1996 and by 2010, was planted on 73% of the total cotton plantings (USDA ERS, 2011). 

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IX. STATEMENT OF GROUNDS UNFAVORABLE

Bayer CropScience knows of no study data and/or observations associated with insectresistant herbicide-tolerant event T303-3 cotton that will result in adverse environmental 

consequences for its introduction. TwinLink Cotton (T304-40 x GHB119), which contains 

the antecedent event T304-40, received nonregulated status in September 2011 (USDA, 

2011). Since the Cry1Ab and PAT proteins in event T303-3 cotton are the same as those 

of the antecedent event, the assessment for T304-40 informs the assessment of T303-3. 

The evidence and data provided in this petition supports the conclusion that event T303-3 

cotton presents a low risk to human health and the environment and does not pose a plant 

pest risk.

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USDA. 2008. Bayer CropScience LP. Petition for determination of nonregulated status for 

insect-resistant and glufosinate ammonium-tolerant cotton: TwinLinkTM Cotton 

(events T304-40 x 

GHB119). http://www.aphis.usda.gov/brs/aphisdocs/08_34001p.pdf (accessed 25 

Jan. 2012).

USDA. 2011. Determination of Nonregulated Status for TwinLinkTM Cotton (Gossypium 

hirsutum). http://www.aphis.usda.gov/brs/aphisdocs/08_34001p_det.pdf 

(accessed 26 Jan. 2012). 

USDA-ERS. 2011. Agricultural biotechnology: Adoption of biotechnology and its 

production 

impacts. http://www.ers.usda.gov/Briefing/Biotechnology/chapter1.htm (accessed 

25 Jan. 2012).

U.S. Food and Drug Administration (US-FDA). 2003. Biotechnology Consultation: Note to 

the File BNF No. 000086. Glufosinate tolerant cotton transformation event 

LLCotton25. June 5, 

2003. http://www.fda.gov/Food/Biotechnology/Submissions/ucm155782.htm

(accessed 26 Jan. 2012). 

Van Deynze, A.E., F.J. Sundstrom, and K.J. Bradford. 2005. Pollen-mediated gene flow in 

California cotton depends on pollinator activity. Crop Science 45:1565–1570.

Wozenski J., and M. Woodburn. 1975. Phytic acid (myoinositol hexaphosphate) and 

phytase activity in four cottonseed protein products. Cereal Chemistry 52:665-669. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 56 of 105
Appendix 1. Field Trial Termination Reports 2001-2006

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 57 of 105
USDA 2001 Termination Report for Bt Cotton

Bayer CropScience LP 

Trials Conducted: State (County)

01-075-08n: MS (Washington)

01-075-18n: MS (Washington)

01-292-01n: PR (Juana Diaz)

Trials Not Conducted: State (County)

01-292-01n: PR (Sabana Grande)

01-312-05n: PR (Juana Diaz, Sabana Grande)

Planting Dates

May 25, 2001 (Washington Co., MS) through December 5, 2001 (Juana Diaz, PR) 

Harvest and Plot Destruction Dates

November 8, 2001 (Washington Co., MS) through June 28, 2002 (Juana Diaz, PR) 

Purpose

Field trials were conducted to test the efficacy as well as breeding of transgenic herbicide-tolerant cotton. 

Transgenic plants contained the bar gene expressing the PAT enzyme, which confers resistance to the 

broad-spectrum herbicide glufosinate-ammonium, as well as the Cry1Ab gene allowing resistance to attack 

by various lepidopteran pest species. 

Observations

Experienced personnel qualified in cotton cultivation performed all plot observations. Recorded 

observations for transgenic and non-transgenic control plots were provided from first square through open 

boll growth stages. 

Plant emergence patterns were uniform and vigorous within both plot types. Germination rates ranged from 

80 to 90% (Washington Co., MS). 

No morphological differences were noted between transgenic and non-transgenic plants. The only in-field 

phenotypic difference observed between the two genotypes was their respective levels of tolerance to 

lepidopteran feeding pressure.

Insect pest species listed for the Washington Co., MS site included bollworm (Helicoverpa zea), plantbugs 

(Lygus lineolaris), and cutoworms (Agrotis upsilon). At the Juana Diaz, PR site only bollworm 

(Helicoverpa zea) was reported. There were no differences recorded in either diversity or density of insect 

pest species found between transgenic and non-transgenic plots. 

A single species of beneficial insect, the ladybug (Hippodamia convergens), was recorded at the 

Washington Co., MS site. At Juana Diaz, PR honeybees (Apis melifera) and aphid lions (Chrysopa sp 

larvae) were recorded. As with the pest species, no differences were noted between species profiles for the 

two plot types. 

No phtyopathology was recorded at either site.

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 58 of 105
Post-Trail Monitoring

Volunteer monitoring revealed post-season plants at the Washington Co., MS sites with less than ten plants 

found on April 17, 2002, and the Juana Diaz, PR site with less than ten plants found on July 24, 2002. 

These were hand-weeded. Clean inspections were established by May 3, 2002 and August 23, 2002, 

respectively.

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 59 of 105
Field Termination Report

USDA ID# 02-058-02n

Page 1 of 3

USDA Field Termination Report

Notification No.: 02-058-02n

Applicant No.: BT-2A-Cotton-MR

Permittee: Aventis CropScience (Now Bayer CropScience LP) 

Research Triangle Park, NC; 919-549-2655 

Regulated Article: Herbicide-tolerant, glufosinate-ammonium-tolerant; 

Insect-resistant, resistant to Lepidopteran pest species, 

Cotton (Gossypium hirsutum) 

Site Release Information: As shown below, one (1) of two (2) sites was planted:

County/Parish/District State or Territory Release Status

Washington MS Planted

Lubbock TX Not Planted

Release information for Washington Co., MS is as 

follows: 

Acreage

Planted

Date

Planted

Germination Data

Transgenic vs. Non-transgenic

Date

Terminated

0.35 5/20/02 % Emergence/Seedling Vigor

70% vs 85% on 5/27/02

Initial Stand Count Percentage

75% vs 90% on 6/4/02

11/26/02

Final Stand Count

1.4 plants per foot vs 1.6 plants 

per foot on 6/28/02

Purpose of Release: The purpose of the release was to test the efficacy of the 

transgenic herbicide-tolerant cotton plants. Transgenic 

plants contained the Cry1Ab gene, allowing resistance

to attack by various lepidopteran pest species. The 

performance of the transgenic cotton with respect to the 

nontransgenic counterpart plant was also evaluated, as 

were the overall agronomic characteristics.

Observations: The test site was inspected twelve (12) times during the 

growing season (5/27/02, 5/31/02, 6/4/02, 6/19/02, 

6/20/02, 6/26/02, 6/28/02, 7/8/02, 7/12/02, 8/1/02, 

8/9/02, 8/12/02) for agronomic growth characteristics 

and disease and insect pest infestation. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 60 of 105
Field Termination Report

USDA ID# 02-058-02n

Page 2 of 3

Observations were recorded from first square through 

open boll growth stages on both the transgenic and 

nontransgenic plants. Transgenic plants did not 

germinate quite as well as the nontransgenic 

counterpart, but both grew vigorously. 

Two (2) species of insect pests were noted: thrips and 

plant bugs. Thrips were seen on 5/31/02, and plant bugs 

were observed on 7/12/02 and 8/1/02. Damage levels 

ranged from slight to moderate. No differences were 

recorded in the diversity of insect pest species found 

between the transgenic and nontransgenic counterpart.

The only beneficial insect observed was the ladybug 

which was seen on 6/19/02, 7/12/02 and 8/12/02. 

No disease susceptibility was noted on the transgenic or 

non-transgenic plants during any of the visits made on 

5/27/02, 6/20/02, 7/8/02 or 11/26/02. 

Results: The only in-field phenotypic differences observed 

between the transgenic and non-transgenic plants were 

in the level of tolerance to lepidopteran feeding pressure 

and tolerance to herbicide (glufosinate-ammonium) 

treatment. 

Plant Disposition: The crop was harvested 11/26/02. Following harvest, 

all remaining plant material was shredded, burned and 

disked under. 

Volunteer Monitoring: The plot area was visually inspected for volunteer 

cotton plants four (4) times during the following 

growing season. No volunteer plants were observed 

during any of the visits.

Weediness Characteristics: No difference in weediness characteristics between the 

transgenic and non-transgenic cotton lines was 

observed. 

Non-Target Organisms: No adverse effect on non-target organisms from either 

the transgenic or nontransgenic plants was observed in 

the trial.

Weather Synopsis: Weather notations indicate the site experienced normal 

climatic conditions during the growing season, but was 

usually wet at harvest time. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 61 of 105
Field Termination Report

USDA ID# 02-058-02n

Page 3 of 3

Containment Measures: Transgenic cotton plants were separated from 

conventional cotton plants by a distance of ~250 feet. A 

40-foot wide perimeter of nontransgenic cotton plants 

bordered the test plot to prevent the flow of pollen from 

transgenic to nontransgenic plants. The border rows 

were destroyed at the conclusion of the trial and were 

monitored as part of the test plot the following growing 

season. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 62 of 105
Field Termination Report

USDA ID# 02-078-01n

Page 1 of 3

USDA Field Termination Report

Notification No.: 02-078-01n

Applicant No.: BT-2B-Cotton-MR

Permittee: Aventis CropScience (Now Bayer CropScience LP) 

Research Triangle Park, NC; 919-549-2655 

Regulated Article: Herbicide-tolerant, glufosinate-ammonium-tolerant; 

Insect-resistant, resistant to Lepidopteran pest species, 

Cotton (Gossypium hirsutum) 

Site Release Information: Release information for Washington Co., MS is as 

follows: 

Acreage

Planted

Date

Planted

Germination Data

Transgenic vs. Non-transgenic

Date

Terminated

0.35 5/20/02 % Emergence/Seedling Vigor

70% vs 85% on 5/27/02

Initial Stand Count Percentage

75% vs 90% on 6/4/02

11/26/02

Final Stand Count

1.4 plants per foot vs 1.6 plants 

per foot on 6/28/02

Purpose of Release: The purpose of the release was to test the efficacy of the 

transgenic herbicide-tolerant cotton plants. Transgenic 

plants contained the Cry1Ab gene, allowing resistance

to attack by various lepidopteran pest species. The 

performance of the transgenic cotton with respect to the 

nontransgenic counterpart plant was also evaluated, as 

were the overall agronomic characteristics.

Observations: The test site was inspected twelve (12) times during the 

growing season (5/27/02, 5/31/02, 6/4/02, 6/19/02, 

6/20/02, 6/26/02, 6/28/02, 7/8/02, 7/12/02, 8/1/02, 

8/9/02, 8/12/02) for agronomic growth characteristics 

and disease and insect pest infestation. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 63 of 105
Field Termination Report

USDA ID# 02-078-01n

Page 2 of 3

Observations were recorded from first square through 

open boll growth stages on both the transgenic and 

nontransgenic plants. Transgenic plants did not 

germinate quite as well as the nontransgenic plants, but 

both grew vigorously. 

Two (2) species of insect pests were noted: thrips and 

plant bugs. Thrips were seen on 5/31/02, and plant bugs 

were observed on 7/12/02 and 8/1/02. Damage levels 

ranged from slight to moderate. No differences were 

recorded in the diversity of insect pest species found 

between the transgenic and nontransgenic counterpart.

The only beneficial insect observed was the ladybug 

which was seen on 6/19/02, 7/12/02 and 8/12/02. 

No disease susceptibility was noted on the transgenic or 

non-transgenic plants during any of the visits made on 

5/27/02, 6/20/02, 7/8/02 or 11/26/02. 

Results: The only in-field phenotypic differences observed 

between the transgenic and non-transgenic plants were 

in the level of tolerance to lepidopteran feeding pressure 

and tolerance to herbicide (glufosinate-ammonium) 

treatment. 

Plant Disposition: The crop was harvested 11/26/02. Following harvest, 

all remaining plant material was shredded, burned and 

disked under. 

Volunteer Monitoring: The plot area was visually inspected for volunteer 

cotton plants four (4) times during the following 

growing season. No volunteer plants were observed 

during any of the visits.

Weediness Characteristics: No difference in weediness characteristics between the 

transgenic and non-transgenic cotton lines was 

observed. 

Non-Target Organisms: No adverse effect on non-target organisms from either 

the transgenic or nontransgenic plants was observed in 

the trial.

Weather Synopsis: Weather notations indicate the site experienced normal 

climatic conditions during the growing season, but was 

usually wet at harvest time. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 64 of 105
Field Termination Report

USDA ID# 02-078-01n

Page 3 of 3

Containment Measures: Transgenic cotton plants were separated from 

conventional cotton plants by a distance of ~250 feet. A 

40-foot wide perimeter of nontransgenic cotton plants 

bordered the test plot to prevent the flow of pollen from 

transgenic to nontransgenic plants. The border rows 

were destroyed at the conclusion of the trial and were 

monitored as part of the test plot the following growing 

season. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 65 of 105
Field Termination Report

USDA ID# 02-261-25n

Page 1 of 3

USDA Field Termination Report

Notification No.: 02-261-25n

Applicant No.: BT-2D-Cotton-MR

Permittee: Bayer CropScience LP

Research Triangle Park, NC; 919-549-2655 

Regulated Article: Herbicide-tolerant, glufosinate-ammonium-tolerant; 

Insect-resistant, resistant to Lepidopteran pest species, 

Cotton (Gossypium hirsutum) 

Site Release Information: Release information for Sabana Grande District, PR is 

as follows: 

Acreage

Planted

Date

Planted

Germination Data

Transgenic vs. Non-transgenic

Date

Terminated

0.22 11/19/02 % Emergence/Seedling Vigor

>50% vs >50% on 11/30/02

Initial Stand Count Percentage

>80% vs >80% on 12/6/02

4/23/03

Final Stand Count Percentage

40-60% vs >80% on 12/20/02

Purpose of Release: The purpose of the release was for breeding and seed 

increase. Transgenic plants contained the Cry1Ab gene, 

allowing resistance to attack by various lepidopteran 

pest species. The performance of the transgenic cotton 

with respect to the nontransgenic counterpart plant was 

also evaluated, as were the overall agronomic 

characteristics.

Observations: The test site was inspected fifteen (15) times during the 

growing season (11/26/02, 11/30/02, 12/6/02, 12/8/03, 

12/10/02, 12/17/02, 12/20/02, 1/2/03, 2/15/03, 2/23/03, 

3/1/03, 3/10/03, 4/5/03, 4/15/03, 4/16/03) for agronomic 

growth characteristics and disease and insect pest 

infestation. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 66 of 105
Field Termination Report

USDA ID# 02-261-25n

Page 2 of 3

Observations were recorded from first square through 

open boll growth stages on both the transgenic and 

nontransgenic plants. Both the transgenic and 

nontransgenic plants germinated well and grew 

vigorously. 

Four (4) species of insect pests were noted: grasshoppers 

(12/6/02), leafminers (12/17/02), aphids (12/20/02), and 

armyworms (1/2/03). Damage levels ranged from slight 

to moderately severe. No differences were recorded in 

the diversity of insect pest species found between the 

transgenic and nontransgenic counterpart.

Two (2) species of beneficial insects were observed: 

cucumber beetle on 12/20/02 and honeybees on 3/1/03. 

No disease susceptibility was noted on the transgenic or 

non-transgenic plants during any of the visits made on 

12/6/02, 2/15/03, 3/10/03 or 4/5/03. 

Results: The only in-field phenotypic differences observed 

between the transgenic and non-transgenic plants were 

in the level of tolerance to lepidopteran feeding pressure 

and tolerance to herbicide (glufosinate-ammonium) 

treatment. 

Plant Disposition: The crop was harvested over two (2) days: 4/16/03 and 

4/17/03. Following harvest, all remaining plant material 

was mechanically cultivated 4/23/03. 

Volunteer Monitoring: The plot area was visually inspected for volunteer 

cotton plants five (5) times during the following 

growing season, until no volunteers had been observed 

for two (2) consecutive post-season monitoring visits.

Post-Season Volunteer Monitoring

Date No. Plants Observed/Stage Method of Destruction

4/30/03 >50 plants/V2 Mechanically Cultivated

5/14/03 >50 plants/V3 Mechanically Cultivated

6/4/03 11 to 50 plants/V2 Mechanically Cultivated

6/24/03 None Field Mechanically Cultivated

7/18/03 None Field Mechanically Cultivated

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 67 of 105
Field Termination Report

USDA ID# 02-261-25n

Page 3 of 3

Weediness Characteristics: There was no evidence of change in characteristics that 

would enhance survival of the glufosinate-ammoniumtolerant transgenic cotton plants as compared to the 

non-transgenic cotton plants. No difference in 

weediness characteristics between the transgenic and 

non-transgenic cotton lines was observed. 

Non-Target Organisms: No adverse effect on non-target organisms from either 

the transgenic or nontransgenic plants was observed in 

the trial.

Weather Synopsis: Weather notations indicate the site experienced typical 

climatic conditions during the growing season. 

Containment Measures: The Sabana Grande test site is a 59-acre farm. The site 

produces no commercial crops. A 40-foot-wide 

perimeter of nontransgenic cotton surrounded the test 

plot to prevent the flow of pollen. At trial conclusion, 

the border rows were not harvested but were destroyed 

along with the remnants of the test plot. The border 

area was monitored (as part of the test plot) for 

volunteer cotton plants during the period that followed. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 68 of 105
Field Termination Report

USDA ID# 03-070-12n

Page 1 of 3

USDA Field Termination Report

Notification No.: 03-070-12n

Applicant No.: BT/HT-3A-Cotton-MR

Permittee: Bayer CropScience LP

Research Triangle Park, NC; 919-549-2655 

Regulated Article: Herbicide-tolerant, Glufosinate-ammonium-tolerant; 

Insect-resistant, resistant to Lepidopteran insect pests, 

Cotton (Gossypium hirsutum) 

Site Release Information: All release sites authorized under this notification were 

planted. Below are acreage amounts, planting dates and 

termination dates for each site:

County/State Acreage

Planted

Date Planted Date

Terminated

Oktibbeha/MS 2.75 5/27/03 10/24/03

Washington/MS 1.2 5/29/03 12/5/03

Lubbock/TX 0.69 5/26/03 & 6/9/03 1/10/04

Purpose of Release: The trials were established to evaluate the efficacy of the 

herbicide-tolerant/insect-resistant cotton plants. 

Transgenic plants contained the cry1Ab gene, allowing 

resistance to attack by various lepidopteran pest species. 

The performance of the transgenic cotton with respect to 

the nontransgenic counterpart was also evaluated, as 

were the overall agronomic characteristics.

Observations: The test sites were visually inspected multiple times 

during the growing season for agronomic growth 

characteristics and disease and insect pest infestation. 

Observations were recorded for the transgenic and 

nontransgenic plants from emergence through harvest.

County or District/ 

State or Territory

Germination Data

Transgenic vs. Non-transgenic

Oktibbeha/MS Mean germination ranged from 55% to

95.25% between the various cotton

lines.

Washington/MS % Emergence/Seedling Vigor

85% vs. 85% on 6/5/03

Initial Stand Count Percentage

90% vs. 90% on 6/13/03

Final Stand Count Percentage

90% vs. 90% on 9/18/03

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 69 of 105
Field Termination Report

USDA ID# 03-070-12n

Page 2 of 3

County or District/ 

State or Territory

Germination Data

Transgenic vs. Non-transgenic

Lubbock/TX % Emergence/Seedling Vigor

65.3% vs. 62% on 7/16/03

Initial Stand Count Percentage

65% vs. 62% on 7/16/03

Final Stand Count Percentage

65% vs. 62% on 8/19/03

In Oktibbeha Co., some plots were infested with H. zea 

and some were not. Each week, for a period of six (6) 

weeks, damage and larvae counts were made.

In general, transgenic cotton plants exhibited normal 

growth and development at each of the locations. 

Experimental treatments of glufosinate-ammonium 

were made. Hail damage necessitated a replant in 

Lubbock Co. 

County/State

Observations/Dates

Fungi/Diseases Insect Pests Beneficial Insects

Oktibbeha/MS None observed 6/11/03, Some plots were infested No notation was made of

7/6/03, 8/6/03 and with H. zea. No record the presence of any 

8/24/03. made of the presence of beneficial insects. 

any other insect species.

Washington/MS None observed 6/5/03, A light infestation of plant Lady beetles were

7/8/03, 7/28/03 and bugs was seen 7/28/03 and observed on 6/5/03,

11/24/03. 8/2/03. 7/8/03 and 8/2/03.

Lubbock/TX None observed 7/16/03, A light infestation of Ladybugs were observed

7/28/03 and 8/15/03. bollworms and leafminers 7/16/03 and 8/15/03, 

were observed 7/38/03. adult hooded beetles 

Aphids were observed were observed 7/28/03, 

10/20/03. and lacewings were seen

8/15/03.

Results: No disease susceptibility or resistance differences were 

observed between the transgenic cotton plants and its 

nontransgenic counterpart. Some phenotypic 

differences were observed between the transgenic and 

nontransgenic plants in terms of the level of tolerance to 

herbicide treatment and susceptibility to lepidopteran 

pests.

Plant Disposition: The Oktibbeha Co. trial was harvested 10/23/03. Stalks 

were cut and disked under the following day. In 

Washington Co., harvest occurred 12/1/03 and 12/2/03. 

Stalks were mowed, and lint and seed burned on 

12/5/03. The Lubbock Co. trial was destroyed 1/10/04 

by burning the plot and disking it under. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 70 of 105
Field Termination Report

USDA ID# 03-070-12n

Page 3 of 3

Volunteer Monitoring: The plot areas were visually inspected for volunteer 

cotton plants during the following growing season. The 

table below summarizes observations made and actions 

taken to eliminate volunteer plants. 

Post-Season Volunteer Monitoring

County/State Date No. Plants Method of Destruction

Observed/Stage

Oktibbeha/MS 5/24/04 None

6/2/04 None

7/14/04 None

7/21/04 None

7/28/04 None

Washington/MS 4/5/04 None

4/13/04 None

4/28/04 None

5/6/04 None

5/14/04 None

Lubbock/TX 4/2/04 None

4/16/04 1 to 10 plants Plants were removed by hand and 

field was mechanically cultivated. 

4/30/04 None

5/13/04 1 to 10 plants Plants were removed by hand and 

herbicide was applied. 

5/26/04 None

6/15/04 None

Weediness Characteristics: There was no evidence of change in characteristics that 

would enhance survival of the transgenic cotton plants 

as compared to the nontransgenic cotton plants. No 

difference in weediness characteristics was observed 

between the transgenic and nontransgenic cotton lines.

Non-Target Organisms: No adverse effect on non-target organisms from either 

the transgenic or nontransgenic plants was observed 

during any of the trials.

Weather Synopsis: The weather in Oktibbeha Co. was noted to be wet.

Conditions for Washington Co. were normal. Lubbock 

Co. was wet and cold early in the season. 

Containment Measures: A 40-foot-wide perimeter of nontransgenic or 

commercial cotton surrounded the test plots to minimize 

pollen flow. The border rows were not harvested but 

destroyed at the conclusion of the trial. The border

areas were monitored along with the actual test plot the 

next growing season for volunteer cotton plants.

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 71 of 105
BT-5A-Cotton-MR 1 05-040-06n

USDA Field Termination Report

USDA Notification Number: 05-040-06n

Applicant Internal Number: BT-5A-Cotton-MR

Applicant: Bayer CropScience LP

Research Triangle Park, NC 27709 

(919) 549-2655

Regulated Article: Gossypium hirsutum; plant incorporated protectant (PIP); tolerant 

to glufosinate-ammonium herbicide

Site Release Information: Trials utilizing this trait were conducted at four (4) locations. A 

fifth site located in Bossier Parish, LA was not planted:

County / State Acreage

Planted

Date

Planted

Date

Terminated Isolation Method

Pinal Co., AZ 0.426 5/27/05 11/25/05 660 ft isolation distance

Gaines Co., TX 0.96 5/9/05 11/7/05 40 ft isolation buffer

Uvalde Co., TX 0.2755 4/22/05 9/23/05 660 ft isolation distance

Wharton Co., TX 0.2355 5/5/05 10/6/05 660 ft isolation distance

Purpose of Release: Trials were established to evaluate the performance of Plant

Incorporated Protectant traits in various cotton varieties. 

Observations:

• Pinal Co., AZ: Observations were made between planting and harvest four times during the 

season. A non-transgenic check was not present to compare the regulated article to, but 

cooperator observed no abnormal increases or decreases in insect, disease, or weed pressure 

given the climate and region the cotton was planted in. Cooperator did indicate that seed 

germination was lower than normal, but this was most likely due to the variety of plant the 

transgenic event was tested in.

• Gaines Co., TX: Observations were made between planting and harvest four times during the 

season. Cooperator observed a difference in the early emergence of the cotton, with the nontransgenic line emerging at 4.88 plants/ft. (81% germination) vs. 2.87 plants/ft. (47% 

germination) for the transgenic lines at the 1-2 leaf stage of growth. Observations taken one 

month later showed that this difference was no longer present, with the transgenic lines having 

caught up to the non-transgenic lines. No other differences in disease, insect pests, or 

phenotypical differences were seen between the transgenic and non-transgenic lines. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 72 of 105
BT-5A-Cotton-MR 2 05-040-06n

• Uvalde Co., TX: Observations were made between planting and harvest four times during the 

season. Cooperator observed differences between transgenic and non-transgenic lines with 

transgenic lines emerging at cotyledon stage at 45% and the non-transgenic counterpart at

7%. This difference was less noticeable within 14 days of the previous observations, but still 

had a stand which was 5-6% greater than the non-transgenic line. Both lines showed equal 

infestations of Texas root rot and Boll Dangle, with an equal mortality rate of plants which 

contracted root rot. Additionally, transgenic lines showed a much lower infestation of cotton 

bollworms, with infestation rates in the non-transgenic lines ranging from 20-500% greater 

infestation than the transgenic varieties. No other differences were noted in weediness 

characteristics, phenotype, or beneficial insect populations. 

• Wharton Co., TX: Observations were made between planting and harvest four times during

the season. Cooperator noticed little to no differences between transgenic and non-transgenic 

lines, with a slight decline in one transformation event of this trait (most likely attributable to the

plant variety). Cooperator did notice that the transgenic plants appeared to be more of an 

“open plant type” but this could not be assigned to either the plant variety or the transgenic 

trait. Bollworm pressure never developed fully enough to evaluate the efficacy of the PIP trait.

Plant Disposition: Plants were harvested between late September and late 

November of 2005. All field sites were destroyed with no seed 

retained. Remaining plant material in the field was cultivated into 

the soil after harvest. All seed sent to cooperators was utilized in 

the planting of these trials.

Volunteer Monitoring: Volunteer monitoring is currently being conducted on the Pinal 

Co., Gaines Co., and Uvalde Co. trial sites. Monitoring is 

scheduled to conclude one year from the dates of harvest. 

Monitoring was discontinued at the Wharton Co. site on 5/19/06 

due to the replant of another regulated article in the same trial 

area. 

Weediness Characteristics: There was no indication of increased weediness characteristics in 

either the transgenic or non-transgenic varieties. 

Non-target Organisms: There was no indication of any adverse effects to non-target 

insect populations or beneficial insects. Various beneficial insect 

populations were present with lacewings, assassin bugs, lady 

beetles, and various coleopteran species observed.

Weather Synopsis:

• Pinal Co., AZ: Reported drier than normal fall season. 

• Gaines Co., TX: Reported as normal for this region of Texas. 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 73 of 105
BT-5A-Cotton-MR 3 05-040-06n

• Uvalde Co., TX: Abnormally dry the entire season, with the month of July being abnormally hot.

• Wharton Co., TX: Abnormally hot all season, with dry conditions existing from June to mid-July.

Containment Measures: All plots, with the exception of the site in Gaines Co., TX, were 

isolated from other sexually compatible species and commercial 

cotton by a distance of at least 660 feet. The Gaines Co. site was 

isolated from the surrounding cotton by an isolation border of at 

least 40 feet. This isolation border was validated by internal audit.

Signature: Date: 6/30/06 

Cynthia A. Allen 

Regulatory Compliance Specialist

Bayer CropScience – Regulatory Affairs BioScience 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 74 of 105
BT-5D-Cotton-MR 1 05-091-08n

USDA Field Termination Report

USDA Notification Number: 05-091-08n

Applicant Internal Number: BT-5D-Cotton-MR

Applicant: Bayer CropScience LP

Research Triangle Park, NC 27709 

(919) 549-2655 

Regulated Article: Gossypuim hirsutum; plant incorporated protectant (PIP); tolerant 

to glufosinate-ammonium herbicide

Site Release Information: Trials utilizing this trait were conducted at six (6) locations:

County / State Acreage

Planted

Date

Planted

Date

Terminated Isolation Method

Crittenden Co., AR 0.24 5/24/05 11/1/05 660 ft. spatial isolation

Jackson Co., AR 0.293 5/26/05 11/4/05 660 ft. spatial isolation

Tift Co., GA 0.29 6/21/05 12/14/05 660 ft. spatial isolation

Tate Co., MS 0.17 5/30/05 10/28/05 660 ft. spatial isolation

Hockley Co., TX 0.27 5/26/05 11/21/05 660 ft. spatial isolation

Wharton Co., TX 0.26 6/8/05 11/14/05 660 ft. spatial isolation

Purpose of Release: Trials were established to evaluate the performance of Plant 

Incorporated Protectant traits in various cotton varieties, and 

generate samples for residue analysis of cotton varieties. 

Observations:

• Crittenden Co., AR: Cooperator noted significant reduction in the germination rate of the 

transgenic plots throughout the trial period, although non-transgenic checks within the plot

were also below average germination indicating possible issues with the evaluated variety. No 

differences were noted in plant diseases, insect pest populations, beneficial insect populations, 

or weediness characteristics. Cooperator did note that transgenic variety initially seemed to 

lag behind the non-transgenic variety in vigor, but later caught up, and was growing normally. 

There was also a 15-25% difference the boll opening between the transgenic and nontransgenic varieties, which the cooperator attributed to the low plant population of the 

transgenic variety. 

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BT-5D-Cotton-MR 2 05-091-08n

• Jackson Co., AR: Cooperator noted significant reduction in the germination rate of the 

transgenic plots throughout the trial period, although non-transgenic checks within the plot

were also below average germination indicating possible issues with the evaluated variety. No 

significant differences were noted in plant diseases, insect pest populations, beneficial insect 

populations, or weediness characteristics. Cooperator did note that transgenic plants 

appeared to be shorter and did not appear as healthy as non-transgenic plants, but mentioned 

this could have been due to the thin plant stand.

• Tift Co., GA: Cooperator noted significant reduction in the germination rate of the transgenic 

plots throughout the trial period, although non-transgenic checks within the plot were also

below average germination indicating possible issues with the evaluated variety. No significant

differences were noted in plant diseases or beneficial insect populations. Cooperator did note 

significant reductions in budworm and bollworm populations in the transgenic variety, and also 

noted a higher percentage of plants with multiple terminals in the non-transgenic. This trend 

was noted in the non-transgenic variety as well, but to a lesser degree, indicating this to be a 

varietial effect, rather than a transgenic one. Cooperator also noted that transgenic variety 

seemed to outperform non-transgenic variety in growth and fruit set. 

• Tate Co., MS: Cooperator noted significant reduction in the germination rate of the transgenic 

plots throughout the trial period, although non-transgenic checks within the plot were also

below average germination indicating possible issues with the evaluated variety. No significant 

differences were noted in plant diseases, insect pest populations, beneficial insect populations, 

physical plant characteristics, or weediness characteristics.

• Hockley Co., TX: Cooperator noted significant reduction in the germination rate of the 

transgenic plots throughout the trial period, although non-transgenic checks within the plot

were also below average germination indicating possible issues with the evaluated variety. No 

significant differences were noted in plant diseases, insect pest populations, beneficial insect 

populations, physical plant characteristics, or weediness characteristics. 

• Wharton Co., TX: Cooperator noted significant reduction in the germination rate of the 

transgenic plots throughout the trial period, although non-transgenic checks within the plot

were also below average germination indicating possible issues with the evaluated variety. No

significant differences were noted in plant diseases, insect pest populations, beneficial insect 

populations. Cooperator did note that transgenic varieties had very low emergence, and did 

not mature normally throughout the season. This resulted in low retention of fruit, increased 

vegetative growth, and showed an increased incidence of hard lock. 

Plant Disposition: For all six locations, plots were harvested and data taken, with 

samples sent to Bayer facility for analysis. All remaining 

harvested material in the field and in the lab was destroyed. 

Unplanted seed which was not utilized by the cooperator was 

either returned to Bayer for storage or destroyed.

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BT-5D-Cotton-MR 3 05-091-08n

Volunteer Monitoring: Volunteer monitoring is ongoing for all trial sites and is scheduled

for completion one year from the date of harvest.

Weediness Characteristics: There was no indication of increased weediness characteristics in 

either the transgenic or non-transgenic varieties for most plots. 

Multiple terminal growth in both varieties in Tift Co., GA was 

noted, along with the stunted development of the transgenic 

variety in Wharton Co., TX. In both instances, this development 

was attributed to the plant variety. 

Non-target Organisms: There was no indication of any adverse effects to non-target 

insect populations or beneficial insects. Various beneficial insect 

populations were present with lacewings, assassin bugs, lady 

beetles, and various coleopteran species observed.

Weather Synopsis:

• Crittenden Co., AR: Below normal rainfall; compensated via irrigation of plots. 

• Jackson Co., AR: Normal weather conditions

• Tift Co., GA: Slightly elevated rainfall amounts in late summer

• Tate Co., MS: Below average rainfall

• Hockley Co., TX: Weather was within normal averages for this region; a hail storm was 

reported early in the season with minimal (<5%) damage to plots. 

• Wharton Co., TX: Plots experienced above average temperatures for this region. Minor plot 

damage resulted from Hurricane Rita on 9/24/05, but an evaluation 

showed that there was no loss of lint or seed from the plot.

Containment Measures: All plots were isolated from other cotton by a distance of at least 

660 ft. from the plot perimeter.

Signature: Date: 6/30/06 

Cynthia A. Allen 

Regulatory Compliance Specialist

Bayer CropScience – Regulatory Affairs BioScience 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 77 of 105
IR-5A-GH-MR 1 05-189-07n

USDA Field Termination Report

USDA Notification Number: 05-189-07n

Applicant Internal Number: IR-5A-GH-MR

Applicant: Bayer CropScience LP

Research Triangle Park, NC 27709 

(919) 549-2655

Regulated Article: Gossypium hirsutum; plant incorporated protectant (PIP); tolerant 

to glufosinate-ammonium herbicide

Site Release Information: Trials utilizing this trait were conducted at one (1) location:

County / State Acreage

Planted

Dates

Planted

Date(s)

Terminated Isolation Method(s)

Sabana Grande, PR 1.015 11/15/05 5/4/06 Isolation border &

physical barrier

Purpose of Release: This trial was established to evaluate the performance of plant 

incorporated protectant traits in cotton varieties. 

Observations: Cooperator did not note any differences between transgenic and 

non-transgenic varieties other than effects of the PIP on bollworm 

populations. No other differences in beneficial insect populations, 

plant diseases, physical plant characteristics, or weediness 

characteristics were reported.

Plant Disposition: Cooperator confirmed that all seed sent by Bayer and not used in 

the creation of the trial was returned. The plot was harvested

5/4/06. All harvested material was sent to the Bayer facility in 

Lubbock, Texas. Residual plant material was incorporated into 

the trial site.

Volunteer Monitoring: Volunteer monitoring is currently being conducted and is 

scheduled for completion one year from the date of harvest. 

Weediness Characteristics: There was no indication of increased weediness characteristics in 

either the transgenic or non-transgenic varieties. 

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IR-5A-GH-MR 2 05-189-07n

Non-target Organisms: There was no indication of any adverse effects to non-target

insect populations or beneficial insects.

Weather Synopsis: Cooperator reported normal growing conditions for this region.

Plot Damage: No damage to plots was reported.

Containment Measures: Plot was contained utilizing a 40 ft isolation border. As a 

secondary measure to prevent gene flow, a nylon/mesh screen 

cage was erected which enclosed the transgenic plants. The 

cage was erected before flowering and remained until harvest. 

Signature: Date: 6/30/06 

Cynthia A. Allen 

Regulatory Compliance Specialist

Bayer CropScience – Regulatory Affairs BioScience 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 79 of 105
Page 1 of 4

USDA Field Termination Report

USDA Notification Number: 06-047-02n Applicant 

Reference Number: IR-6A-GH-MR Applicant:

Bayer CropScience LP

Research Triangle Park, NC 27709

Regulated Article: Gossypium hirsutum (cotton)

Site Release Information: Trials utilizing this trait were conducted in fourteen (14) locations.

County / State Acreage

Planted Date Planted Date Terminated Isolation Method

Bossier/ LA 0.70 5/31/06 11/14/06 Isolation Border Rows

Madison/ LA 1.28 6/05/06 12/05/06 Isolation Border Rows

Coahoma/ MS 1.28 6/06/06 10/06/06 Isolation Border Rows

Oktibbeha/ MS 0.648 5/30/06 10/10/06 Isolation Border Rows

Washington/ MS

(2-locations) 2.34 5/30/06 &

6/06/06

11/28/06 &

12/18/06 Isolation Border Rows

Durham & Halifax/ 

NC 0 - - Trials were not initiated

Dillon/ SC (2-locations) 3.88 6/02/06 &

5/30/06

12/08/06 &

12/11/06 Isolation Border Rows

Gaines/ TX 0.73 5/26/06 12/05/06 Isolation Border Rows

Lubbock/ TX (1) 0.165 5/24/06 12/14/06 Isolation Border Rows

Lubbock/ TX (2) 0 - - Trial was not initiated

Uvalde/ TX

(2-locations) 1.12 5/25/06 10/30/06 &

10/03/06 Isolation Border Rows

Wharton/ TX

(2-locations) 1.2989 6/08/06 11/14/06 Isolation Border Rows

Observations: listing of characteristics between the transgenic and non-transgenic:

1. Bossier Pa., LA

 Cooperator noted no unusual characteristics.

 Seedling disease was discovered in both transgenic and non-transgenic.

 No differences in plant disease, insect pest or beneficial insect populations.

 Non-transgenic slightly taller than transgenic.

 Transgenic had more “hook-billed” bolls.

2. Madison Pa., LA: 

 No unusual characteristics.

 No differences in emergence, maturity, flowering, plant disease, insect pest

population, weediness or beneficial insect populations.

IR-6A-GH-MR 06-047-02n 

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Page 80 of 105
IR-6A-GH-MR 2 of 4 06-047-02n

3. Coahoma Co., MS: 

 Approximately 120 days after planting the cotton, drift from a nearby field the

cooperator was spraying, caused the site to prematurely defoliate into a total 

loss of yield. Therefore, crop was not harvested and was destroyed with a

mechanical shredder.

 Prior to defoliation there were no significant differences between the

transgenic and non-transgenic. Observations were considered normal.

4. Oktibbeha Co., MS: 

 Plots were artificially infested with cotton boll worms.

 Non-transgenic plots were grown without artificial infestation of the cotton boll

worms.

 No significant differences were noted in plant diseases, insect pest population,

beneficial insect populations, weediness or physical plant characteristics.

5. Washington Co., MS: 

 Cooperator noted that there were no significant differences in plant diseases,

insect pest and beneficial insect populations.

 Everything appeared normal between the transgenic and non-transgenic from

the first true-leaf stage to maturity.

6. Washington Co., MS: 

 Cooperator noted that during maturity the non-transgenic had a better fruit

load than the transgenic.

 No significant differences were noted in plant diseases, insect pest

populations, and beneficial insect populations.

 No significant differences in emergence, squaring, blooming or maturity.

 A delay in harvesting due to an unusual cool and wet autumn

7. Durham Co., NC:

 Study cancelled due to business reasons therefore, planting was never

initiated.

8. Halifax Co., NC:

 Study was cancelled due to receiving seed too late.

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IR-6A-GH-MR 3 of 4 06-047-02n

9. Dillon Co., SC (1): 

 Cooperator did note that transgenic varieties had severe delayed maturity and

parrot beak non-opened bolls at maturity.

 Agronomic performance deemed as unacceptable. Efficacy data was biased

based on poor crop performance for other physiological aspects of the line 

tested.

 No significant differences were noted in plant diseases, insect pest

populations or beneficial insect populations.

10. Dillon Co., SC (2): 

 Cooperator noted delayed maturity, parrot beak underdeveloped and nonopening bolls.

 Agronomic performance deemed as unacceptable.

 Genetic Effects are unacceptable and many bolls never opened.

 Efficacy data was biased based on poor crop performance

 There were no significant differences in plant diseases, insect pest, and

beneficial insect populations.

11. Gaines Co., TX: 

 Cooperator noted no significant differences in plant diseases, insect pest, and

beneficial insect populations.

 It was noted that all other visual observations were normal.

 There was some hail but it did not cause damage or loss to the plot.

12. Lubbock Co., TX (1): 

 Cooperator noted no significant differences in plant diseases, insect pest, and

beneficial insect populations.

 Observations show no significant differences between the transgenic and nontransgenic plots.

13. Lubbock Co., TX (2): 

 Study cancelled due to business reasons therefore, planting was never

initiated

14. Uvalde Co., TX (1): 

 No significant differences were noted in plant diseases, insect pest or

beneficial populations.

 Observations show that the transgenic plot had delayed maturity and the

cotton bolls were unacceptably small.

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IR-6A-GH-MR 4 of 4 06-047-02n

15. Uvalde Co., TX (2): 

 No significant differences were noted in plant diseases, insect pest or

beneficial populations

 It was noted by the cooperator that the boll size for the transgenic was far

superior to the non-transgenic

16. Wharton Co., TX (1) & (2): 

 Cooperator noted early boll opening for transgenic and non transgenic.

 The transgenic plot showed resistance to bollworm, budworm infestations.

 No significant differences were noted in plant diseases, insect pest or

beneficial insect populations.

 No unusual growth patterns.

Plant Disposition: Plots were harvested and data taken, with samples sent to Bayer 

facility for analysis. All remaining harvested material in the field 

was destroyed. Unplanted seed which was not utilized by the 

cooperator was either returned to Bayer for storage, or destroyed.

Volunteer Monitoring: Volunteer monitoring is currently being conducted on all trial sites, 

and scheduled to conclude one year from the termination date.

Weediness Characteristics: There was no indication of increased weediness characteristics in 

either the transgenic or non-transgenic plots.

Non-target Organisms: There was no indication of any adverse effects to non-target 

insect populations or beneficial insects. Various beneficial insect 

populations were present with lacewings, assassin bugs, lady 

bugs, and various coleopteran species observed.

Weather: Weather for all sites was either normal or hot and dry. However, 

Washington County, Mississippi autumn season was unusually 

cool and wet causing a delay in harvesting and Wharton County, 

Texas (2), July and October was extremely wet.

Signature: Date: 9/19/07

Regina J. Hayes

Regulatory Compliance Specialist

Bayer CropScience – Regulatory Affairs BioScience

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 83 of 105
IR-6D-GH-MR 1 of 3 06-068-03n

USDA Field Termination Report

USDA Notification Number: 06-068-03n

Applicant Reference Number: IR-6D-GH-MR 

Applicant: Bayer CropScience LP

Research Triangle Park, NC 27709

Regulated Article: Gossypium hirsutum (cotton)

Site Release Information: Trials utilizing this trait were conducted in ten (10) locations.

County / State Acreage

Planted Date Planted DateTerminated Isolation Method

St. Landry/ LA 1.84 6/12/06 11/16/06 Isolation Border Rows

Tate/ MS (2 locations) 1.46 5/31/06 11/ 10/06 Isolation Border Rows

Washington/ MS 1.49 6/08/06 12/02/06 Isolation Border Rows

Hockley/ TX 

(2 locations) 1.92 5/30/06 & 6/06/06 12/18/06 Isolation Border Rows

Uvalde/ TX 

(2 locations) 2.85 5/30/06 & 6/13/06 11/10/06 Isolation Border Rows

Wharton/ TX 

(2 locations) 4.61 6/13/06 & 6/14/06 11/14/06 &

11/20/06 Isolation Border Rows

Observations: listing of characteristics between the transgenic and non-transgenic:

1. St. Landry Co., LA: 

 No unusual characteristics.

 No difference in emergence, both plots had early flowering, boll set and boll

opening with some boll rot.

 No differences in plant disease, insect pest or beneficial insect populations

2. Tate Co., MS: 

 No significant differences were noted in plant diseases, insect pest and

beneficial insect populations.

 Cooperator did note that maturity of bolls were significantly slower in the

transgenic plot as compared to the non-transgenic.

 Seedling Vigor in the transgenic plot was approximately 40% of the nontransgenic.

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IR-6D-GH-MR 2 of 3 06-068-03n

3. Washington Co., MS: 

 Cooperator stated that over-all the plots are similar and there are no

significant differences.

 The plots yield was poor and the transgenic appears to have slightly less fruit

set. 

 Misshaped bolls are present in transgenic and non-transgenic plots

 The hot dry weather seemed to stress the plants and as a result fruit shedding

occurred.

 Regimen A has set more fruit than Regimen B or C.

 Regimens B and C had poor yields.

 No significant differences were noted in plant diseases, insect pest, and

beneficial insect populations.

4. Hockley Co., TX:

 Cooperator stated there were no difference in emergence and no significant

differences in plant diseases, insect pest populations, beneficial insect

populations, emergence, flowering, boll set and boll opening.

5. Uvalde Co., TX: 

 No significant differences were noted in plant diseases, insect pest, beneficial

insect and wild life populations.

 Due to low yield (15%) emergence) a decision was made to re- plant.

Replanting took place two (2) weeks later. During the re-planting, previously 

emerged plants were not removed.

 It was noted that the transgenic was less tolerant of whiteflies than the nontransgenic. Possible, two-week difference in planting date may have been the 

cause.

 It was observed that the transgenic cotton was very light and fluffy, there was

no worm presence but the whiteflies made up for the lack of worm presence.

6. Wharton Co., TX: 

 Cooperator noted no unusual characteristics/growth patterns.

 No significant differences were noted in plant diseases, insect pest or

beneficial insect populations.

 Early bloom for transgenic and non-transgenic.

 Transgenic emergence was lower than expected and the non-transgenic had

good emergence.

 Bolls in transgenic were smaller than normal

Plant Disposition: Plots were harvested and data taken, with samples sent to Bayer 

facilities for analyses. All remaining harvested material in the field 

was destroyed. Unplanted seed which was not utilized by the 

cooperator was either returned to Bayer for storage, or destroyed.

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IR-6D-GH-MR 3 of 3 06-068-03n

Volunteer Monitoring: Volunteer monitoring is currently being conducted on all trial sites, 

and scheduled to conclude one year from the termination date.

Weediness Characteristics: There was no indication of increased weediness characteristics in 

either the transgenic or non-transgenic plots.

Non-target Organisms: There was no indication of any adverse effects to non-target 

insect populations or beneficial insects. Various beneficial insect 

populations were present with lacewings, assassin bugs, lady 

beetles, and various coleopteran species observed.

Weather: Weather for all sites were either normal or hot and dry. Wharton 

County, Texas experienced an extremely wet season for the 

months of July and October.

Signature: Date: 9/19/07 

Regina J. Hayes

Regulatory Compliance Specialist

Bayer CropScience – Regulatory Affairs BioScience

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 86 of 105
Appendix 2. Materials and Methods

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Page 87 of 105
2.A. Insert Characterization

 

Southern analyses were conducted to characterize the DNA insertion in event T303-3 

cotton. Genomic DNA was isolated from transgenic and non-transgenic plants and these 

samples were subjected to Southern analyses using the different components of the 

transgenic cassette (3’me1, cry1Ab, P35S, bar, 3’nos, 5’e1) as well as the complete TDNA fragment as probes.

Test Items, Reference Items, and Standards 

Test Item

BC2F2 of FM966 event T303-3 was grown in the greenhouse and leaf tissues were 

harvested from individual plants for genomic DNA extraction.

Control Cotton Material

Non-transgenic cotton variety FM966 seed were planted in the greenhouse and leaf 

tissues were harvested from individual plants for genomic DNA extraction.

Reference Material

Plasmid DNA from pTDL004 was prepared from E.coli strain MC1061. 

λ DNA digested with PstI (Fermentas, Burlington, Ontario, Canada) was used as the 

molecular weight marker on the agarose gels.

Genomic DNA Extraction

Total genomic DNA was prepared from leaf material harvested from event T303-3 cotton 

and from leaf material of non-transgenic cotton variety FM966. The frozen tissue 

samples were pulverized and the DNA was extracted using milli-Q (MQ) water and 

chloroform:isoamylalcohol. To remove all undissolved particles present in the DNA 

samples, the final DNA samples were centrifuged. All samples had large, viscous pellets. 

The supernatant was removed to a clean tube and the pellets were dissolved in MQ 

water overnight. The concentration of the DNA samples was determined. 

Restriction Digests of Total Genomic DNA

For each Southern blot, nine aliquots of 10 μg of event T303-3 genomic DNA were 

digested with the following restriction enzymes: BamHI, BglII, SacI, KpnI, NcoI, PvuI, 

XbaI, EcoRV and NotI. Two aliquots of 10 μg genomic DNA from the non-transgenic 

control were digested with the NotI restriction enzyme. The total reaction volume was 50 

μl and the digests were incubated overnight at 37o

C.

Five μg of plasmid pTDL004 DNA were digested with NotI. The total reaction volume 

was 50 μl and the incubation was performed for approximately three hours at 37°

C. 

Plasmid DNA digestion was checked by agarose gel electrophoresis and the four 

expected fragments (6167bp, 4915bp, 1532bp and 1290bp) were observed. The 

concentration of the digested plasmid DNA was determined to be 95.33 ng/μl.

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Page 88 of 105
Gel Electrophoresis of Digested DNA Samples

Four identical agarose gels were prepared. An appropriate dilution of the NotI digested 

pTDL004 sample was made. Assuming a single copy integration of the transgene into 

the cotton genome, 10 μg of homozygous genomic DNA corresponds to ~ 30.9 pg of 

pTDL004 plasmid DNA. The amount representing approximately one plasmid copy per 

genome was added to 10μg of digested non-transgenic DNA. This served as the positive 

control and was used to show that the hybridizations were performed under conditions 

allowing hybridization of the probes with the target sequences.

After addition of the loading dye, each digest was loaded onto the gel. λ DNA digested 

with PstI was included as the molecular weight standard. Following electrophoresis, the 

separated DNA fragments were transferred from the agarose gel to a Hybond-N+ 

membrane.

DNA Probe Hybridization

Probe templates each containing one of the genetic elements of plasmid pTDL004 and 

the complete T-DNA probe were prepared by PCR. Identical PCR reactions were pooled 

and following volume reduction by evaporation, the different PCR products were loaded 

on an agarose gel and purified by gel extraction. To determine the quality of the probe 

templates, samples of each were loaded on an agarose gel and electrophoresed. The 

determined molecular weight of each probe template fit with the expected fragment sizes. 

The concentration of the different probe templates was determined by comparing band 

intensities of the purified probe templates with band intensities of a low DNA mass ladder 

(Invitrogen Corp., Carlsbad, CA). The templates were stored in the freezer.

For hybridization, the probe templates were labeled with [α32P]. Visualization of the 

hybridizing fragments was achieved via exposure of a BioMax MR film to the hybridized 

membrane.

Stripping of Probes

Membranes were stripped of the radioactively-labeled probes to prepare the blot for 

subsequent re-hybridization to additional probes.

Determination of Hybridization Fragment Sizes

Lengths of all hybridization fragments were determined by comparing them with the 

molecular weight marker. A semi-logarithmic standard curve was constructed using the λ

DNA marker with the y-axis being the distance between the gel loading slot and the 

different fragments of digested marker DNA and the sizes of the fragments of the DNA 

marker on the x-axis. The length of each hybridization fragment was determined by 

measuring the migration distance of the fragment and extrapolating it to the standard 

curve.

2.B. Confirmation of Absence of Vector Backbone

Southern analyses were conducted to confirm the absence of vector backbone from 

plasmid pTDL004. Genomic DNA was isolated from event T303-3 plant material and 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION T303-3 Cotton USDA Petition 

Page 89 of 105
probed with overlapping elements of the vector backbone. Samples were then probed 

with fragments of the T-DNA (p35S and cry1Ab) as a positive control.

Test Items, Reference Items, and Standards

Test Item

BC2F3 of FM966 event T303-3 was grown in the greenhouse and leaf tissues were 

harvested from individual plants for genomic DNA extraction.

Control Cotton Material

Non-transgenic cotton variety FM966 seed were planted in the greenhouse and leaf 

tissues were harvested from individual plants for genomic DNA extraction.

Reference Material

Plasmid DNA from pTDL004 was prepared from E.coli strain MC1061. 

λ DNA digested with PstI (Fermentas, Burlington, Ontario, Canada) was used as the 

molecular weight marker on the agarose gels.

Genomic DNA Extraction

Total genomic DNA was prepared from leaf material harvested from event T303-3 cotton 

and from leaf material of non-transgenic cotton variety FM966. 

Restriction Digests of Total Genomic DNA

Seven samples each of 10 μg of T303-3 genomic DNA were digested with NotI or EcoRV 

restriction enzymes and 28 samples each of 10 μg non-transgenic control genomic DNA 

were digested with the NotI restriction enzyme. All digestions were performed in a total 

reaction volume of 40 μl. The digests were incubated overnight at 37°C.

Five μg of pTDL004 plasmid DNA was digested with NotI. The total reaction volume was 

50 μl. Incubation was performed overnight at 37°C. To check that the plasmid DNA was 

totally digested, 200 ng of the digest were loaded on a 1% TAE horizontal submarine 

agarose gel. All fragments separated as expected (6167 bp, 4915 bp, 1532 bp and 1290 

bp). The concentration of the NotI –digested plasmid DNA was determined at 98.56 ng/μl 

by measuring the fluorescence of the Quant-iTTM PicoGreen® dsDNA reagent.

Gel Electrophoresis of Digested DNA Samples

Seven identical agarose gels were prepared. An appropriate dilution of the NotI digested 

plasmid pTDL004 sample was made. Assuming a single copy integration of the 

transgene into the cotton genome, 10 μg of homozygous genomic DNA corresponds to ~ 

30.9 pg of pTDL004 plasmid DNA. The amount representing approximately 0.1, 1 and 10 

plasmid copies per genome was added to 10 μg of digested non-transgenic DNA. These 

preparations served as the positive controls and were used to show that the 

hybridizations were performed under conditions allowing hybridization of the probes with 

the target sequences.

Each digest was loaded onto the gel in its appropriate well. λ DNA digested with PstI was 

included as the molecular weight standard. 

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Page 90 of 105
Following electrophoresis, the separated DNA fragments were transferred from the 

agarose gel to a Hybond-N+ membrane.

DNA Probe Hybridization with Vector Backbone Probe Templates 

The vector backbone probes were prepared by [α32P] labeling of probe templates PT002, 

PT003, PT005, PT007, PT008, PT009 and PT010. Each of the seven membranes was 

hybridized with a different [α32P] labeled probe. Visualization of the hybridizing fragments 

was achieved via exposure of a BioMax MR film to the hybridized membrane. 

Following the stripping of vector backbone probes from the membranes, each membrane 

was re-hybridized with the T-DNA probe. The T-DNA probe was prepared by [α-32P]

labeling. Visualization of the hybridizing fragments was also achieved via exposure of a 

BioMax MR film to the hybridized membrane. 

Determination of Hybridization Fragment Sizes

Lengths of all hybridization fragments were determined by comparing them with the 

molecular weight marker. A semi-logarithmic standard curve was constructed using the λ

DNA marker with the y-axis being the distance between the gel loading slot and the 

different fragments of digested marker DNA and the sizes of the fragments of the DNA 

marker on the x-axis. The length of each hybridization fragment is determined by 

measuring the migration distance of the fragment and extrapolating it to the standard 

curve.

2.C. Protein (Cry1Ab and PAT) Expression in Raw Agricultural Commodity 

Fuzzy seed (ginned seed but not yet de-linted) from event T303-3 cotton were analyzed 

for the expression of Cry1Ab and PAT proteins using enzyme-linked immunosorbent

assays (ELISAs).

Field production of Seed Samples

Generation T4 event T303-3 cotton seed and non-transgenic Coker 315 cotton seed were 

grown in six trials in the U.S. All trials were managed according to regional commercial 

production practices. Samples of fuzzy seed were collected from ginned harvested 

samples from each plot at each trial site.

Reference Materials

Cry1Ab and PAT proteins, produced in E.coli, were used as standards for the ELISA 

methods.

Processing of Fuzzy Seed Samples

The fuzzy seed was processed by mechanical de-linting. The de-linted seed was 

mechanically cracked in a roller mill and the hull material was separated from the kernel. 

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Protein Extraction from Samples

The sample materials were prepared by either grinding in a blender pre-chilled with dry 

ice or by grinding with a mortar and pestle. The ground sample was mixed with 

extraction buffer and shaken for 30 min at 4°

C. Insoluble material was removed by 

centrifugation. The supernatants were then analyzed by the specific ELISAs for the 

presence of the Cry1Ab and PAT proteins.

Determination of Cry1Ab and PAT Protein Concentrations

The amount of Cry1Ab and PAT protein in the sample extracts were determined by 

commercial ELISA kits (EnviroLogix, Inc., Portland, ME). All assays resulted in color 

development in the last step, which was measured by optical density of each 

microtiterplate well at 450nm. Softmax ProTM software (Molecular Devices, Version 4.0) 

was used to derive the concentration of immunoreactive Cry1Ab and PAT proteins. The 

optical density (OD) values were adjusted for the buffer blank and then any background 

due to the matrix was subtracted, using the average value from two wells containing nontransgenic extracts, assayed on the same microtiter plate. The OD corrected for the 

buffer blank and the non-transgenic background was converted to the protein analyte 

concentration using the standard curve from the same microtiter plate.

2.D. Protein Characterization

Because a full equivalence study (USDA, 2008) was done on the Cry1Ab protein isolated 

from TwinLink Cotton (where event T304-40, the antecedent organism, is one of the 

parents in the TwinLink breeding stack), only an SDS-PAGE analysis was done in this 

study to compare Cry1Ab protein produced in event T303-3 to that produced in E. coli 

and event T304-40. 

Test Items, Reference Items, and Standards

Protein expressed in planta

Event T303-3 and event T304-40 plants were grown in the greenhouse and leaves were 

harvested into plastic bags and placed on dry ice. Leaf samples were processed in a 

Waring blender pre-chilled with dry ice, with small amounts of dry ice added during the 

blending process to ensure samples remained frozen. Ground samples were stored in a 

freezer at approximately -20°C overnight to allow for dry ice dissipation prior to extraction 

of protein. 

The Cry1Ab proteins were extracted by mixing ground plant leaves with extraction buffer 

at a ratio of 25 g of ground leaves to 100 mL of extraction buffer in 5 oz specimen

containers. The 850 mL of extraction buffer contained 150 mM NaCl and 100 mM sodium

phosphate (pH 7.2), 0.85 g of polyvinylpyrrolidinone (40,000 Mol. Wt), 1 mM PMSF, 1 

mM benzamidine/caproic acid and 850 μg of leupeptin/antipain.

After addition of the extraction buffer to the ground leaf material, the 5 oz container with

extraction buffer and ground leaf material was continuously inverted on a rotary rocker at

~30 rpm for 20 minutes at room temperature. The extract was filtered through Miracloth®

and the filtrate vacuum filtered through a 0.45 μm cellulose acetate membrane.

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The Cry1Ab antibody affinity columns used for the purification were prepared using

approximately 750 μL of crude rabbit polyclonal antibody serum raised against the 

Cry1Ab protein. The Cry1Ab antibodies were covalently attached to individual Pierce

AminoLink® Plus immobilization columns. 

Protein Expressed by E. coli 

Trypsin-digested Cry1Ab protein (batch no. 25.05.99.70, purity > 85%) expressed by E. 

coli was provided by Bayer BioScience N.V. Molecular and Biochemical Analytical 

Services- Protein Characterization, Zwijnaarde, Belgium. 

Thrombin-engineered Cry1Ab protein from E.coli (batch no. Min1418-1, purity > 85%) 

was provided by Bayer BioScience N.V. Molecular and Biochemical Analytical ServicesProtein Characterization, Zwijnaarde, Belgium. 

Molecular Weight Markers

Molecular weight markers (Invitrogen BenchMark, Cat# 10747) comprised of a series of 

recombinant proteins of known molecular weight.

SDS-PAGE

The Cry1Ab protein from E. coli and the Cry1Ab protein isolated from events T303-3 and 

T304-40 cotton leaves were analyzed by SDS-PAGE. The protein from the plants and the 

corresponding protein from E. coli were denatured and analyzed by electrophoresis on a 

polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R-250 to visualize 

the protein bands.

SDS-PAGE analysis was also conducted as described above on trypsin-digested Cry1Ab 

protein from E. coli, the thrombin-engineered Cry1Ab protein from E. coli, and Cry1Ab 

protein isolated from the leaves of event T304-40.

The electrophoretic mobility on a polyacrylamide gel of recombinant proteins of known 

molecular weight (Molecular Weight Markers) was utilized to generate a standard curve 

of electrophoretic mobility for each of the SDS-PAGE analyses runs. The standard 

curves were utilized to determine the molecular weights of the Cry1Ab proteins from E. 

coli (trypsin-digested and thrombin-engineered), event T304-40, and event T303-3. 

2.E. Composition Analysis

Samples were analyzed to assess the nutrient and anti-nutrient composition of event 

T303-3 cotton grain and compare it to that of the non-transgenic control. 

Field production of Seed Samples

Generation T4 event T303-3 and non-transgenic cotton seed (Coker 315) were grown in 

six trials in primary cotton growing regions of the U.S in 2005. All trials were managed 

according to regional commercial production practices. Each trial consisted of nine plots. 

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Six plots were event T303-3 and three were non-transgenic Coker 315. Of the six event 

T303-3 plots, three were treated twice with a tank mix of glufosinate ammonium herbicide 

and ammonium sulfate at 0.52 lb ai/A (equivalent to 40 oz Ignite® Herbicide/A) and 3.0 

lb/A, respectively. Samples of fuzzy seed were collected from each plot (i.e., three 

samples T303-3 sprayed, three samples T303-3 nonsprayed, three samples nontransgenic Coker 315) at each trial site and shipped frozen to Bayer CropScience, RTP, 

NC.

Composition Analysis of Raw Agricultural Commodity

Subsamples of frozen ginned seed were sent to two 3rd party labs for composition 

analysis, Eurofins Scientific (Des Moines, IA) and Covance Laboratories (Madison, WI). 

All parameters with the exception of cyclopropenoid fatty acid content were performed by 

Eurofins Scientific. 

Methods utilized by laboratories for each parameter analyzed are shown in Table 21. 

Statistical Methods

Analysis was conducted by analyzing for differences between the three treatments (i.e., 

event T303-3 sprayed, event T303-3 unsprayed, non-transgenic Coker 315) for 55 

different components. These components were:

• Proximate and fiber compounds: moisture, protein, crude fat, ash, total 

carbohydrates, ADF, and NDF

• Minerals: Calcium, Phosphorus, Potassium, Magnesium, Iron, and Zinc 

• Tocopherols: alpha-, beta-,gamma-, and delta tocopherol, total tocopherol 

(Vitamin E) 

• Anti-nutrients: free gossypol, “-“ and “+” gossypol, total gossypol, phytic acid 

• Amino acids: alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, 

histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, 

threonine, tryptophan, tyrosine, and valine

• Fatty acids: myristic (C14:0), palmitic (C16:0), stearic C18:0), arachidic (C20:0), 

behenic (C22:0), lignoceric (C24:0), palmitoleic (C16:1), oleic (C18:1), linoleic 

(C18:2), linolenic (C18:3) 

• Cyclopropenoid fatty acids: malvic acid, sterculic acid and dihydrosterculic acid

Statistical analyses included descriptive statistics (i.e., mean value, standard deviation, 

min/max values), over all analysis (i.e., ANOVA by treatment, site, and respective 

interaction terms), and by site analysis (i.e., ANOVA by treatment where significant 

interactions [p<0.05] by site were detected, followed by t-tests). Level of significance was 

fixed 0.05 (two-sided). 

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Table 21. Analytical methods used for compositional analysis

Parameter Laboratory Method

Moisture Eurofins Scientific AOAC 930.15

Crude fat Eurofins Scientific AOAC 920.39

Crude protein Eurofins Scientific AOCS 990.03

Ash Eurofins Scientific AOAC 942.05

Acid Detergent Fiber Eurofins Scientific ANKOM ADF 05/03

Neutral Detergent Fiber Eurofins Scientific ANKOM NDF 05/03

Carbohydrates Eurofins Scientific

21CFR - Difference 

between 100 and the sum 

of crude protein, fat, 

moisture and ash

Ca Eurofins Scientific AOAC 984.27 and 985.01

P Eurofins Scientific AOAC 984.27 and 985.01

K Eurofins Scientific AOAC 984.27 and 985.01

Fe Eurofins Scientific AOAC 984.27 and 985.01

Mg Eurofins Scientific AOAC 984.27 and 985.01

Zn Eurofins Scientific AOAC 984.27 and 985.01

Tocopherols Eurofins Scientific AOAC 971.30 with HPLC 

Quantitation

Amino Acids Eurofins Scientific AOAC 982.30 Sec. D, F

Fatty Acids Eurofins Scientific AOCS Ce 1e-91 (Capillary 

GC)

Phytic Acid Eurofins Scientific Analytical Biochemistry 77, 

536-539 (1977)

Gossypol (total) Eurofins Scientific AOCS Ba 8-78 (1983, 

reapproved 1997)

Gossypol (free) Eurofins Scientific

Method version of AOCS Ba 

7-58 (reapproved 1997) and 

JAOCS vol. 59, no. 12, pp. 

546-549 (Dec. 1982)

(Modified)

Cyclopropenoid Fatty Acids Covance Laboratories Covance method procedure 

MP-CPFA-MA (HPLC)

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Appendix 3. Characterization of Event T303-3 

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3.A. Insert Characterization 

Genomic DNA isolated from event T303-3 cotton was digested with one of the following 

restriction enzymes: BamHI, BglII, SacI, KpnI, NcoI, PvuI, XbaI, EcoRV, and NotI. Nontransgenic genomic DNA digested with NotI was used as a negative control; nontransgenic genomic DNA supplemented with one copy of pTDL004 and digested with 

NotI was used as a positive control. The resulting DNA fragments were analyzed by 

Southern blot with six different probes, corresponding to the different genetic elements 

contained in the T-DNA from plasmid pTDL004. Probe information is presented in Table 

22. An overview of the obtained Southern blot results is present in Figure 11. 

The results of the Southern blot analysis demonstrate that the inserted transgenic 

sequence in event T303-3 consists of one complete copy of the cry1Ab and bar genes 

with the associated regulatory sequences required for expression.

Table 22. Event T303-3 insert verification – probe information

Probe Template ID Genetic Element Size Probe Template (bp)

PT020-1 3’me1 probe 859

PT021-1 cry1Ab probe 1822

PT022-1 P35S3 probe 801

PT023-1 bar probe 425

PT024-1 3’nos probe 214

PT025-1 Complete T-DNA probe 4929

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Figure 11. Event T303-3 insert verification – Southern blot results

3’me1 probe cry1Ab probe

P35S3 probe bar probe

Loading Sequence- All Probes

Lane 2: G. hirsutum event T303-3 – BamHI digested

Lane 3: G. hirsutum event T303-3 – BglII digested

Lane 4: G. hirsutum event T303-3 – SacI digested

Lane 5: G. hirsutum event T303-3 – KpnI digested

Lane 6: G. hirsutum event T303-3 – NcoI digested

Lane 7: G. hirsutum event T303-3 – PvuI digested

Lane 8: G. hirsutum event T303-3 – XbaI digested

Lane 9: G. hirsutum event T303-3 – EcoRV digested

Lane 10: G. hirsutum event T303-3 – NotI digested

Lane 11: G. hirsutum wild type – NotI digested

Lane 12: G. hirsutum wild type + 1 copy pTDL004 –NotI 

digested

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Figure 11 (continued). Event T303-3 insert verification – Southern blot results

3’nos probe

Complete T-DNA probe

Loading Sequence- All Probes

Lane 2: G. hirsutum event T303-3 – BamHI digested

Lane 3: G. hirsutum event T303-3 – BglII digested

Lane 4: G. hirsutum event T303-3 – SacI digested

Lane 5: G. hirsutum event T303-3 – KpnI digested

Lane 6: G. hirsutum event T303-3 – NcoI digested

Lane 7: G. hirsutum event T303-3 – PvuI digested

Lane 8: G. hirsutum event T303-3 – XbaI digested

Lane 9: G. hirsutum event T303-3 – EcoRV digested

Lane 10: G. hirsutum event T303-3 – NotI digested

Lane 11: G. hirsutum wild type – NotI digested

Lane 12: G. hirsutum wild type + 1 copy pTDL004 –NotI 

digested

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3.B. Confirmation of Absence of Vector Backbone 

Genomic DNA isolated from event T303-3 cotton was digested with restriction enzymes 

EcoRV and NotI. Non-transgenic genomic DNA digested with NotI was used as a 

negative control; non-transgenic genomic DNA supplemented with 0.1, 1, and 10 copies 

of plasmid pTDL004 and digested with NotI was used as a positive control. The resulting 

DNA fragments were analyzed by Southern blot with seven overlapping probes, 

corresponding to the different genetic elements contained in the vector backbone of 

plasmid pTDL004 as well as the complete T-DNA probe. Probe information is presented 

in Table 23. 

Table 24 and 25 summarizes the expected and obtained hybridization fragments using 

vector backbone probes and T-DNA probe, respectively. 

The results of the Southern blot analysis confirm than event T303-3 contains no elements 

of the pTDL004 vector backbone.

Table 23. Event T303-3 vector backbone analysis – probe information

Probe Template ID Genetic Element Position in 

pTDL004

Size Probe 

Template (bp)

PT007-1 Vector Backbone 

Probe bp 5135 to 6480 1346

PT002-1 Vector Backbone 

Probe bp 5981 to 8019 2039

PT003-1 Vector Backbone 

Probe bp 7474 to 9461 1988

PT008-1 Vector Backbone 

Probe bp 8794 to 10860 2067

PT009-1 Vector Backbone 

Probe bp 10277 to 11629 1353

PT010-1 Vector Backbone 

Probe bp 10931 to 12526 1596

PT005-1 Vector Backbone 

Probe bp 12019 to 13879 1861

PT011-1 T-DNA probe

(P35S + cry1Ab) bp 1619 to 4187 2492

 

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Table 24. Expected and obtained hybridization fragments with overlapping vector backbone probe templates

Fragment sizes are provided in bp

*Only 85 bp overlap

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Table 25. Expected and obtained hybridization fragments using T-DNA probe

Fragment sizes are provided in bp

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3.C. Composition Analysis

Fuzzy seed was harvested from six field trials. Each trial consisted of three replications 

of event T303-3 not sprayed, event T303-3 sprayed and the non-transgenic Coker 315. 

Fuzzy seed samples were processed and analyzed for proximate, amino acid, fatty acid,

cyclopropenoid fatty acid, mineral, tocopherol, and anti-nutrient composition. Data for 

composition analysis is presented in Table 26, 27, 28 and 29. 

Table 26. Amino acid composition in cottonseed of event T303-3 

Total Amino Acids 

% Dry matter

Reference 

ranges a NonTransgenic

Transgenic

Not sprayed

Transgenic

Sprayed

Mean ± SD Mean ± SD Mean ± SD

Alanine 0.94 ± 0.08 0.99 ± 0.07 1.00 ± 0.06 0.42 - 1.51

Arginine 2.48 ± 0.33 2.62 ± 0.28 2.65 ± 0.22 1.05 - 4.40

Aspartic acid 2.26 ± 0.21 2.30 ± 0.21 2.33 ± 0.17 1.00 - 3.55

Cystine 0.34 ± 0.04 0.36 ± 0.03 0.37 ± 0.03 0.16 - 0.86

Glutamic acid 4.64 ± 0.55 4.91 ± 0.47 4.97 ± 0.38 1.96 - 8.16

Glycine 0.95 ± 0.09 0.99 ± 0.08 1.00 ± 0.06 0.44 - 1.58

Histidine 0.63 ± 0.07 0.67 ± 0.06 0.67 ± 0.05 0.31 - 1.03

Isoleucine 0.70 ± 0.06 0.73 ± 0.07 0.75 ± 0.05 0.35 - 1.17

Leucine 1.33 ± 0.13 1.40 ± 0.12 1.42 ± 0.10 0.63 - 2.23

Lysine 1.11 ± 0.16 1.23 ± 0.15 1.18 ± 0.10 0.52 - 1.65

Methionine 0.38 ± 0.04 0.41 ± 0.03 0.41 ± 0.03 0.15 - 0.54

Phenylalanine 1.22 ± 0.14 1.28 ± 0.13 1.30 ± 0.10 0.54 - 2.03

Proline 0.91 ± 0.11 0.95 ± 0.09 0.97 ± 0.11 0.41 - 1.39

Serine 1.02 ± 0.10 1.08 ± 0.09 1.09 ± 0.07 0.50 - 1.63

Threonine 0.75 ± 0.06 0.79 ± 0.06 0.80 ± 0.04 0.34 - 1.21

Tryptophan 0.28 ± 0.03 0.29 ± 0.02 0.30 ± 0.02 0.10 - 0.49

Tyrosine 0.56 ± 0.06 0.59 ± 0.06 0.61 ± 0.05 0.32 - 1.17

Valine 0.97 ± 0.10 1.02 ± 0.10 1.03 ± 0.08 0.45 - 1.67

Data represent an average of three replicate samples at six field test sites. aReference ranges compiled from OECD (2004), ILSI (2007), Lawhon et al. (1976), and Bertrand et al. (2005)

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Table 27. Fatty acid composition in cottonseed of event T303-3 

Total fatty acids

% Relative

NonTransgeni

Transgenic

Not sprayed

Transgenic

Sprayed Reference rangesa 

Mean ± SD Mean ± SD Mean ± SD

Saturated

Myristic C 14:0 0.70 ± 0.12 0.62 ± 0.07 0.61 ± 0.06 0.53 - 1.17

Palmitic C16:0 23.21 ± 1.38 22.43 ± 1.05 22.34 ± 1.07 21.1 - 29.9

StearicC18:0 2.42 ± 0.11 2.52 ± 0.12 2.51 ± 0.13 2.15 - 3.4

Arachidic C20:0 0.30 ± 0.02 0.28 ± 0.03 0.28 ± 0.02 0 - 0.48

Behenic C22:0 0.17 ± 0.05 0.15 ± 0.01 0.15 ± 0.01 0 - 0.27

Lignoceric C24:0 0.12 ± 0.02 0.11 ± 0.01 0.11 ± 0.01 0 - 0.30

Sum saturated 26.92 26.11 26.00 24.1 - 35.5

Mono-unsaturated

Palmitoleic C16:1 0.51 ± 0.04 0.48 ± 0.04 0.48 ± 0.04 0.46 - 0.86

Oleic C18:1 14.80 ± 0.77 14.61 ± 0.55 14.61 ± 0.50 13.4 - 22.0

Sum mono-unsaturated 15.31 15.09 15.09 13.9 - 22.9

Poly-unsaturated

Linoleic C18:2 56.20 ± 1.71 57.35 ± 1.62 57.47 ± 1.49 36.3 - 64.0

Linolenic C18:3 0.58 ± 0.14 0.56 ± 0.04 0.55 ± 0.04 < 0.10 - 0.62

Sum poly-unsaturated 56.78 57.91 58.02 36.3 - 64.3

Sum of total fatty acids 99.01 99.11 99.11 -

Data represent an average of three replicate samples at six field test sites aReference ranges compiled from OECD (2004), ILSI (2007), Bertrand et al. (2005), Berberich et al. (1996),

and Nida et al. (1996)

Table 28. Cyclopropenoid fatty acid composition in cottonseed of event T303-3 

Parameter

Based on dry

Reference 

rangesa

NonTransgeniTransgenic

Not sprayed

Transgenic

Sprayed

Mean ± SD Mean ± SD Mean ± SD

Malvalic acid %rel. 0.389 ± 0.076 0.361 ± 0.093 0.354 ± 0.088 0.17 - 1.5

Sterculic acid %rel. 0.331 ± 0.045 0.306 ± 0.054 0.324 ± 0.065 0.12 - 0.92

Dihydrosterculic acid

%rel. 0.149 ± 0.020 0.150 ± 0.015 0.146 ± 0.016 0.11 - 0.50 

Data represent an average of three replicate samples at six field test sites. aReference ranges compiled from OECD (2004), ILSI (2007), Calhoun et al. (1995), Berberich et al. (1996), 

Nida et al. (1996), Phelps et al. (1965), and Wozenski and Woodburn (1975)

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Table 29. Mineral and tocopherol composition in cottonseed of event T303-3 

Parameter

Based on dry matter

Reference 

rangesa NonTransgenic

Transgenic

Not sprayed

Transgenic

Sprayed

Mean ± SD Mean ± SD Mean ± SD

Calcium % 0.15 ± 0.02 0.24 ± 0.05 0.27 ± 0.09 0.09 – 0.33

Phosphorus % 0.62 ± 0.08 0.65 ± 0.07 0.66 ± 0.09 0.31 – 0.86

Potassium % 1.18 ± 0.11 1.20 ± 0.06 1.21 ± 0.07 0.96 – 1.42

Magnesium % 0.38 ± 0.03 0.41 ± 0.03 0.41 ± 0.03 0.27 – 0.49

Iron % 0.0061 ± 0.0015 0.0063 ± 0.0015 0.0072 ± 0.0021 0.0023-0.016

Zinc mg/kg 30.26 ± 3.40 37.04 ± 12.81 35.45 ± 5.01 17.8 – 63.0

αTocopherol mg/kg 128.39 ± 25.47 115.31 ± 17.57 124.56 ± 13.41 16 - 245

β Tocopherol mg/kg < 6.00 < 6.00 < 6.00 0 – 10

γTocopherol mg/kg 49.00 ± 18.40 49.22 ± 14.50 58.33 ± 13.61 16 – 271

δ Tocopherol mg/kg < 6.00 < 6.00 < 6.00 0 – 10

Total

Tocopherol (vitamin E)b 177.53 ± 32.31 164.79 ± 27.02 182.56 ± 20.61 32 – 536

Data represent an average of three replicate samples at six field test sites. aReference ranges compiled from OECD (2004), ILSI (2007), USCA (1982), Calhoun et al. (1995), Lundquist

(1995) and FAO/WHO (2001) bVitamin E as the sum of all tocopherols in mg/kg dm

 

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